Brassica INDEHISCENT1 sequences

ABSTRACT

The present application provides  Brassica  INDEHISCENT1 (BIND) sequences.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present patent application claims benefit of priority to U.S.Provisional Patent Application No. 60/608,967, filed on Jun. 18, 2004,which is incorporated by reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with Government support under National ScienceFoundation Grant number IBN-9985530. The Government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Rapeseed is one of the most important oilseed crops after soybeans andcottonseed, representing 10% of the world oilseed production in 1990.Rapeseed contains 40% oil, which is pressed from the seed, leaving ahigh-protein seed meal of value for animal feed and nitrogen fertilizer.Rapeseed oil, also known as canola oil, is a valuable product,representing the fourth most commonly traded vegetable oil in the world.

Unfortunately, the yield of seed from rapeseed and related plants islimited by pod dehiscence, which is a process that occurs late in fruitdevelopment whereby the pod is opened and the enclosed seeds released.Degradation and separation of cell walls along a discrete layer of cellsdividing the two halves of the pod, termed the “dehiscence zone,” resultin separation of the two halves of the pod and release of the containedseeds. The dehiscence zone is a region of only one to three cells inwidth that extends along the entire length of the valve/replum boundary(Meakin and Roberts, J. Exp. Botany 41:995-1002 (1990)). As the cells inthe dehiscence zone separate from one another, the valves detach fromthe replum, allowing seeds to be dispersed. Seed “shattering,” wherebyseeds are prematurely shed through dehiscence before the crop can beharvested, is a significant problem faced by commercial seed producersand represents a loss of income to the industry. Adverse weatherconditions can exacerbate the process of dehiscence, resulting ingreater than 50% loss of seed yield.

The fruit, a complex structure unique to flowering plants, mediates thematuration and dispersal of seeds. In most flowering plants, the fruitconsists of the pericarp, which is derived from the ovary wall, and theseeds, which develop from fertilized ovules. Arabidopsis, which istypical of the more than 3000 species of the Brassicaceae, producesfruit in which the two carpel valves (ovary walls) are joined to thereplum, a visible suture that divides the two carpels.

The plant hormone ethylene is produced by developing seeds and appearsto be an important regulator of the dehiscence process. One line ofevidence supporting a role for ethylene in regulation of dehiscencecomes from studies of fruit ripening, which, like fruit dehiscence, is aprocess involving the breakdown of cell wall material. In fruitripening, ethylene acts in part by activating cell wall degradingenzymes such as polygalacturonase (Theologis et al., Develop. Genetics14:282-295 (1993)). Moreover, in genetically modified tomato plants inwhich the ethylene response is blocked, such as transgenic tomato plantsexpressing antisense polygalacturonase, there is a significant delay infruit ripening (Lanahan et al., The Plant Cell 6:521-530 (1994); Smithet al., Nature 334:724-726 (1988)).

In dehiscence, ultrastructural changes that culminate in degradation ofthe middle lamella of dehiscence zone cell walls weaken rapeseed podsand eventually lead to pod shatter. As in fruit ripening, hydrolyticenzymes including polygalacturonases play a role in this programmedbreakdown. For example, in oilseed rape, a specificendo-polygalacturonase, RDPG1, is upregulated and expressed exclusivelyin the dehiscence zone late in pod development (Petersen et al., PlantMol. Biol. 31:517-527 (1996), which is incorporated herein byreference). Ethylene may regulate the activity of hydrolytic enzymesinvolved in the process of dehiscence as it does in fruit ripening(Meakin and Roberts, J. Exp. Botany 41:1003-1011 (1990), which isincorporated herein by reference). Yet, until now, the proteins thatcontrol the process of dehiscence, such as those regulating the relevanthydrolytic enzymes, have eluded identification.

Attempts to solve the problem of pod shatter and early fruit dehiscenceover the past 20 years have focused on the breeding of shatter-resistantvarieties. However, these plant hybrids are frequently sterile and losefavorable characteristics that must be regained by backcrossing, whichis both time-consuming and laborious. Other strategies to alleviate podshattering include the use of chemicals such as pod sealants ormechanical techniques such as swathing to reduce wind-stimulatedshattering. To date, however, a simple method for producing geneticallymodified plants that do not open and release their seeds prematurely hasnot been described.

Thus, a need exists for identifying genes that regulate the dehiscenceprocess and for developing genetically modified plant varieties in whichthe natural seed dispersal process is delayed. The present inventionsatisfies this need and provides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acids comprising apolynucleotide encoding a polypeptide at least 90% identical to SEQ IDNO:9 or SEQ ID NO:10. In some embodiments, the polypeptide comprises SEQID NO:9. In some embodiments, the polypeptide comprises SEQ ID NO:10. Insome embodiments, the polynucleotide comprises SEQ ID NO:7. In someembodiments, the polynucleotide comprises SEQ ID NO:8. In someembodiments, the polynucleotide comprises SEQ ID NO:11. In someembodiments, the polynucleotide comprises SEQ ID NO:12.

The present invention also provides expression cassettes comprising apromoter operably linked to a polynucleotide, or a complement thereof.In some embodiments, the polynucleotide is at least 90% identical to atleast 200 contiguous nucleotides of SEQ ID NO:7 or SEQ ID NO:8. In someembodiments, the sequence comprises SEQ ID NO:9. In some embodiments,the sequence comprises SEQ ID NO:10. In some embodiments, thepolynucleotide is at least 90% identical to a nucleotide sequenceencoding SEQ ID NO:9. In some embodiments, the polynucleotide comprisesa nucleotide sequence encoding SEQ ID NO:9. In some embodiments, thepolynucleotide is at least 90% identical to a nucleotide sequenceencoding SEQ ID NO:10. In some embodiments, the polynucleotide comprisesa nucleotide sequence encoding SEQ ID NO:10. In some embodiments, thepromoter is constitutive. In some embodiments, the promoter is tissuespecific. In some embodiments, the promoter is a dehiscence zonespecific promoter. In some embodiments, the polynucleotide is operablylinked to the promoter in the antisense orientation. In someembodiments, the polynucleotide is operably linked to the promoter inthe sense orientation.

The present invention also provides plants comprising a recombinantexpression cassette, the expression cassette comprising a promoteroperably linked to a polynucleotide, or a complement thereof. In someembodiments, the polynucleotide comprises a nucleotide sequence at least90% identical to at least 200 contiguous nucleotides of a sequenceencoding SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, the sequencecomprises SEQ ID NO:9. In some embodiments, the sequence comprises SEQID NO:10. In some embodiments, the polynucleotide is at least 90%identical to a nucleotide sequence encoding SEQ ID NO:9. In someembodiments, the polynucleotide comprises a nucleotide sequence encodingSEQ ID NO:9. In some embodiments, the polynucleotide is at least 90%identical to a nucleotide sequence encoding SEQ ID NO:10. In someembodiments, the polynucleotide comprises a nucleotide sequence encodingSEQ ID NO:10. In some embodiments, the promoter is constitutive. In someembodiments, the promoter is tissue specific. In some embodiments, thepromoter is a dehiscence zone specific promoter. In some embodiments,the polynucleotide is operably linked to the promoter in the antisenseorientation. In some embodiments, the polynucleotide is operably linkedto the promoter in the sense orientation. In some embodiments,lignification is reduced in valve margin cells of the plant. In someembodiments, lignification is enhanced in the plant. In someembodiments, the plant is a Brassica species. In some embodiments, theplant is characterized by delayed seed dehiscence compared to a plantnot comprising the expression cassette.

The present invention also provides methods of delaying fruit dehiscencein a plant. In some embodiments, the methods comprise suppressingexpression of an IND1 nucleic acid in the plant by introducing into theplant a recombinant expression cassette comprising a promoter operablylinked to a polynucleotide, or a complement thereof, wherein thepolynucleotide comprises a nucleotide sequence at least 90% identical toat least 200 contiguous nucleotides of a sequence encoding SEQ ID NO:9or SEQ ID NO:10; and selecting a plant with delayed fruit dehiscencecompared to a plant in which the expression cassette has not beenintroduced. In some embodiments, the sequence comprises SEQ ID NO:9. Insome embodiments, the sequence comprises SEQ ID NO:10. In someembodiments, the polynucleotide is at least 90% identical to anucleotide sequence encoding SEQ ID NO:9. In some embodiments, thepolynucleotide comprises a nucleotide sequence encoding SEQ ID NO:9. Insome embodiments, the polynucleotide is at least 90% identical to anucleotide sequence encoding SEQ ID NO:10. In some embodiments, thepolynucleotide comprises a nucleotide sequence encoding SEQ ID NO:10. Insome embodiments, the promoter is constitutive. In some embodiments, thepromoter is tissue specific. In some embodiments, the promoter is adehiscence zone specific promoter. In some embodiments, thepolynucleotide is operably linked to the promoter in the antisenseorientation. In some embodiments, the polynucleotide is operably linkedto the promoter in the sense orientation. In some embodiments, therecombinant expression cassette is introduced into the plant usingAgrobacterium. In some embodiments, the plant is a Brassica species.

Definitions

The terms “nucleic acid” and “polynucleotide” are used synonymously andrefer to a single or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids orpolynucleotides may also include modified nucleotides that permitcorrect read through by a polymerase and do not alter expression of apolypeptide encoded by that nucleic acid. “Polynucleotide sequence” or“nucleic acid sequence” may include both the sense and antisense strandsof a nucleic acid as either individual single strands or in the duplex.It includes, but is not limited to, self-replicating plasmids,chromosomal sequences, and infectious polymers of DNA or RNA.

The phrase “nucleic acid sequence encoding” refers to a nucleic acidthat codes for an amino acid sequence of at least 5 contiguous aminoacids within one reading frame. The amino acid need not necessarily beexpressed when introduced into a cell or other expression system, butmay merely be determinable based on the genetic code. For example, thesequence ATGATGGAGCATCAT encodes MMEHH. Thus, a polynucleotide mayencode a polypeptide sequence that comprises a stop codon or contains achanged frame so long as at least 5 contiguous amino acids within onereading frame. The nucleic acid sequences may include both the DNAstrand sequence that is transcribed into RNA and the RNA sequence thatis translated into protein. The nucleic acid sequences include both thefull length nucleic acid sequences as well as fragments from the fulllength sequences. It should be further understood that the sequenceincludes the degenerate codons of the native sequence or sequences whichmay be introduced to provide codon preference in a specific host cell.

The term “promoter” or “regulatory element” refers to a region orsequence determinants located upstream or downstream from the start oftranscription and which are involved in recognition and binding of RNApolymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Such promoters need not be of plant origin, for example,promoters derived from plant viruses, such as the CaMV35S promoter, canbe used in the present invention.

As used herein, the term “dehiscence zone-selective regulatory element”refers to a nucleotide sequence that, when operatively linked to anucleic acid molecule, confers selective expression upon the operativelylinked nucleic acid molecule in a limited number of plant tissues,including the valve margin or dehiscence zone. The valve margin is thefuture site of the dehiscence zone and encompasses the margins of theouter replum as well as valve cells adjacent to the outer replum. Thedehiscence zone, which develops in the region of the valve margin,refers to the group of cells that separate during the process ofdehiscence, allowing valves to come apart from the replum and theenclosed seeds to be released. Thus, a dehiscence zone-selectiveregulatory element, as defined herein, confers selective expression inthe mature dehiscence zone, or confers selective expression in the valvemargin, which marks the future site of the dehiscence zone.—

A dehiscence zone-selective regulatory element can confer specificexpression exclusively in cells of the valve margin or dehiscence zoneor can confer selective expression in a limited number of plant celltypes including cells of the valve margin or dehiscence zone. ASHATTERPROOF1 or SHATTERPROOF2 (SHP1 and SHP2, previously designated asAGL1 and AGL5, repectively) regulatory element, for example, whichconfers selective expression in ovules and placenta as well as in thedehiscence zone, is a dehiscence zone-selective regulatory element asdefined herein. Similarly, an IND regulatory element also confersselective expression in the dehiscence zone. A dehiscence zone-selectiveregulatory element generally is distinguished from other regulatoryelements by conferring selective expression in the valve margin ordehiscence zone without conferring expression throughout the adjacentcarpel valves.

It is understood that limited modifications can be made withoutdestroying the biological function of a regulatory element and that suchlimited modifications can result in dehiscence zone-selective regulatoryelements that have substantially equivalent or enhanced function ascompared to a wild type IND regulatory element. These modifications canbe deliberate, as through site-directed mutagenesis, or can beaccidental such as through mutation in hosts harboring the regulatoryelement. All such modified nucleotide sequences are included in thedefinition of a dehiscence zone-selective regulatory element as long asthe ability to confer selective expression in the valve margin ordehiscence zone is substantially retained.

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g. leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g. bracts, sepals, petals, stamens, carpels,anthers and ovules), seed (including embryo, endosperm, and seed coat)and fruit (the mature ovary), plant tissue (e.g. vascular tissue, groundtissue, and the like) and cells (e.g. guard cells, egg cells, trichomesand the like), and progeny of same. The class of plants that can be usedin the method of the invention is generally as broad as the class ofhigher and lower plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), gymnosperms,ferns, and multicellular algae. It includes plants of a variety ofploidy levels, including aneuploid, polyploid, diploid, haploid andhemizygous.

The term “seed plant” means an angiosperm or gymnosperm. An angiospermis a seed-bearing plant whose seeds are borne in a mature ovary (fruit).An angiosperm commonly is recognized as a flowering plant. Angiospermsare divided into two broad classes based on the number of cotyledons,which are seed leaves that generally store or absorb food. Thus, amonocotyledonous angiosperm is an angiosperm having a single cotyledon,whereas a dicotyledonous angiosperm is an angiosperm having twocotyledons. A variety of angiosperms are known including, for example,oilseed plants, leguminous plants, fruit-bearing plants, ornamentalflowers, cereal plants and hardwood trees, which general classes are notnecessarily exclusive. The skilled artisan will recognize that themethods of the invention can be practiced using these or otherangiosperms, as desired. A gymnosperm is a seed-bearing plant with seedsnot enclosed in an ovary.

The phrase “host cell” refers to a cell from any organism. Exemplaryhost cells are derived from plants, bacteria, yeast, fungi, insects orother animals. Methods for introducing polynucleotide sequences intovarious types of host cells are well known in the art.

The term “delayed,” as used herein in reference to the timing of seeddispersal in a fruit produced by a non-naturally occurring plant of theinvention, means a statistically significantly later time of seeddispersal as compared to the time seeds normally are dispersed from acorresponding plant at the same developmental stage expressingnaturally-occurring levels of IND. Thus, the term “delayed” is usedbroadly to encompass both seed dispersal that is significantly postponedas compared to the seed dispersal in a corresponding plant, and to seeddispersal that is completely precluded, such that fruits never releasetheir seeds unless there is human or other intervention.

It is recognized that there can be natural variation of the time of seeddispersal within a plant species or variety. However, a “delay” in thetime of seed dispersal in a non-naturally occurring plant of theinvention readily can be identified by sampling a population of thenon-naturally occurring plants and determining that the normaldistribution of seed dispersal times is significantly later, on average,than the normal distribution of seed dispersal times in a population ofthe corresponding plant species or variety that does not contain anexogenous IND polynucleotide. Thus, production of non-naturallyoccurring plants of the invention provides a means to skew the normaldistribution of the time of seed dispersal from pollination, such thatseeds are dispersed, on average, at least about 1%, 2%, 5%, 10%, 30%,50%, 100%, 200% or 500% later than in the corresponding plant speciesthat does not contain an exogenous nucleic acid molecule encoding an INDgene product.

The term “suppressed” or “decreased” encompasses the absence of INDprotein in a plant, as well as protein expression that is present butreduced as compared to the level of IND protein expression in a wildtype plant. Furthermore, the term suppressed refers to IND proteinexpression that is reduced throughout the entire domain of INDexpression, or to expression that is reduced in some part of the INDexpression domain, provided that the resulting plant is characterized bydelayed seed dispersal. The term “suppressed” also encompasses an amountof IND protein that is equivalent to wild type IND expression, but wherethe IND protein has a reduced level of activity. As discussed above, INDeach contain a conserved an basic HLH domain; point mutations or grossdeletions within the HLH domain that reduce the DNA-binding activity ofIND can reduce or destroy the activity of IND and, therefore, “suppress”IND expression as defined herein. One skilled in the art will recognizethat, preferably, IND expression is essentially absent in the valvemargin of a plant or the IND protein is essentially non-functional.

“Increased” or “enhanced” IND activity or expression of a IND generefers to an augmented change in IND activity. Examples of suchincreased activity or expression include the following. IND activity orexpression of the IND gene is increased above the level of that inwild-type, non-transgenic control plants (i.e. the quantity of NDactivity or expression of the IND gene is increased). IND activity orexpression of the IND gene is in an organ, tissue or cell where it isnot normally detected in wild-type, non-transgenic control plants (i.e.spatial distribution of ND activity or expression of the IND gene isincreased). IND activity or expression is increased when IND activity orexpression of the IND gene is present in an organ, tissue or cell for alonger period than in a wild-type, non-transgenic controls (i.e.duration of IND activity or expression of the IND gene is increased).

A polynucleotide sequence is “heterologous to” a second polynucleotidesequence if it originates from a foreign species, or, if from the samespecies, is modified by human action from its original form. Forexample, a promoter operably linked to a heterologous coding sequencerefers to a coding sequence from a species different from that fromwhich the promoter was derived, or, if from the same species, a codingsequence which is different from any naturally occurring allelicvariants.

A polynucleotide “exogenous to” an individual plant is a polynucleotidewhich is introduced into the plant, or a predecessor generation of theplant, by any means other than by a sexual cross. An exogenous nucleicacid molecule can have a naturally occurring or non-naturally occurringnucleotide sequence and can be a heterologous nucleic acid moleculederived from a different plant species than the plant into which thenucleic acid molecule is introduced or can be a nucleic acid moleculederived from the same plant species as the plant into which it isintroduced. Examples of means by which this can be accomplished aredescribed below, and include Agrobacterium-mediated transformation,biolistic methods, electroporation, in planta techniques, and the like.

An “IND polynucleotide” is a nucleic acid sequence substantially similarto SEQ ID NO:1, 7, 8, 11, or 12 or that encodes a bHLH polypeptide thatis substantially similar to SEQ ID NO:2, 9 or 10. IND polypeptides willgenerally have an alanine at site 9 of the basic region of the protein(e.g., amino acid position 129 of SEQ ID NO:2, position 140 of SEQ IDNO:9 and position 112 of SEQ ID NO:10) and generally does not comprise aPAS domain (Nambu (1991) Cell 67:1157-1167; Wilk, R. (1996) Genes Dev.10:93-102). IND polynucleotides may comprise (or consist of) a codingregion of about 50 to about 10,000 or more nucleotides, sometimes fromabout 100 to about 3,000 nucleotides and sometimes from about 200 toabout 600 nucleotides, which hybridizes to SEQ ID NO:1, 7 or 8 understringent conditions (as defined below), or which encodes an INDpolypeptide or fragment of at least 15 amino acids thereof. INDpolynucleotides can also be identified by their ability to hybridizeunder low stringency conditions (e.g., Tm ˜40° C.) to nucleic acidprobes having the sequence of SEQ ID NO:1, 7, 8, 11, or 12. SEQ ID NO:1,7, 8, 11, or 12 are examples of IND polynucleotides.

A “promoter from a IND gene” or “IND promoter” will typically be about500 to about 3000 nucleotides in length, usually from about 750 to 2750.Exemplary promoter sequences are shown as SEQ ID NO:3 and SEQ ID NO:4.SEQ ID NO:3 represents the 5′ untranslated region of the IND and SEQ IDNO:4 represents the 3′ untranslated region of IND. A IND promoter canalso be identified by its ability to direct expression in the valvemargin of fruit. In particular, the IND promoter directs expression atthe valve margin of developing gynoecium just prior to fertilization(stage 13) through the maturation of the fruit (stage 17). The promoterdoes not provide significant expression in leaf tissue.

An “IND polypeptide” is an amino acid sequence that is substantiallysimilar to SEQ ID NOs:2, 9, or 10, or a fragment thereof. Active INDpolypeptides generally have an alanine at site 9 of the basic region ofthe protein (e.g., amino acid position 129 of SEQ ID NO:2) and do notcomprise a PAS domain (Nambu (1991) Cell 67:1157-1167; Wilk, R. (1996)Genes Dev. 10:93-102) Full-length IND polypeptides are characterized bythe presence of an basic helix-loop-helix (HLH) domain which bindspecific polynucleotide sequences. For instance amino acid residuesISDDPQTVVARRRRERISEKIRILKRIVPGGAKMDTASMLDEAIRYTKFLK represent the HLHdomain of the polypeptide shown in SEQ ID NO:2. The HLH domain is knownin the art and is shared by other transcription factors includinguncharacterized sequences represented by GenBank accession numberE1283552 and 2262147 and the gene product, PIF3 (Ni et al. Cell 95:657(1998)). The HLH domain of IND is therefore a DNA binding domain.

As used herein, a homolog or ortholog of a particular IND gene (e.g.,SEQ ID NO:1) is a second gene in the same plant type or in a differentplant type, which has a polynucleotide sequence of at least 50contiguous nucleotides which are substantially identical (determined asdescribed below) to a sequence in the first gene. It is believed that,in general, homologs or orthologs share a common evolutionary past.

A “polynucleotide sequence from” a particular gene is a subsequence orfull length polynucleotide sequence of an IND gene which, when presentin a transgenic plant, has the desired effect. For example, one effectis inhibition of expression of the endogenous gene driving expression ofan heterologous polynucleotide.

The term “reproductive tissues” as used herein includes fruit, ovules,seeds, pollen, pistols, flowers, or any embryonic tissue.

An “expression cassette” refers to a nucleic acid construct, which whenintroduced into a host cell, results in transcription and/or translationof a RNA or polypeptide, respectively. Antisense constructs or senseconstructs that are not or cannot be translated are expressly includedby this definition.

In the case of both expression of transgenes and inhibition ofendogenous genes (e.g., by antisense, or sense suppression) one of skillwill recognize that the inserted polynucleotide sequence need not beidentical and may be “substantially identical” to a sequence of the genefrom which it was derived. As explained below, these variants arespecifically covered by this term.

In the case where the inserted polynucleotide sequence is transcribedand translated to produce a functional polypeptide, one of skill willrecognize that because of codon degeneracy a number of polynucleotidesequences will encode the same polypeptide. These variants arespecifically covered by the term “polynucleotide sequence from” aparticular valve-margin gene, such as IND. In addition, the termspecifically includes sequences (e.g., full length sequences)substantially identical (determined as described below) with a IND genesequence and that encode proteins that retain the function of a INDpolypeptide.

In the case of polynucleotides used to inhibit expression of anendogenous gene, the introduced sequence need not be perfectly identicalto a sequence of the target endogenous gene. The introducedpolynucleotide sequence will typically be at least substantiallyidentical (as determined below) to the target endogenous sequence.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The term “complementary to” is used herein to mean thatthe sequence is complementary to all or a portion of a referencepolynucleotide sequence.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman Add. APL. Math. 2:482(1981), by the homology alignment algorithm of Needle man and Wunsch J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, BLAST,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 25% sequenceidentity. Alternatively, percent identity can be any integer from 25% to100%. Exemplary embodiments include at least: 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. compared to areference sequence using the programs described herein; preferably BLASTusing standard parameters, as described below. Accordingly, INDsequences of the invention include nucleic acid sequences that havesubstantial identity to SEQ ID NO:1, 7, 8, 11 or 12. IND sequences ofthe invention also include polypeptide sequences having substantialidentify to SEQ ID NO:2, 9 or 10. One of skill will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 40%. Preferredpercent identity of polypeptides can be any integer from 40% to 100%,e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 99%, an sometimes at least 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74% and 75%. Polypeptides which are“substantially similar” share sequences as noted above except thatresidue positions which are not identical may differ by conservativeamino acid changes. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other, or a third nucleic acid,under stringent conditions. Stringent conditions are sequence dependentand will be different in different circumstances. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe.Typically, stringent conditions will be those in which the saltconcentration is about 0.02 molar at pH 7 and the temperature is atleast about 60° C.

In the present invention, mRNA encoded by IND genes of the invention canbe identified in Northern blots under stringent conditions using cDNAsof the invention or fragments of at least about 100 nucleotides. For thepurposes of this disclosure, stringent conditions for such RNA-DNAhybridizations are those which include at least one wash in 0.2×SSC at63° C. for 20 minutes, or equivalent conditions. Genomic DNA or cDNAcomprising genes of the invention can be identified using the same cDNAs(or fragments of at least about 100 nucleotides) under stringentconditions, which for purposes of this disclosure, include at least onewash (usually 2) in 0.2×SSC at a temperature of at least about 50° C.,usually about 55° C., for 20 minutes, or equivalent conditions.

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state and may be in either a dry or aqueoussolution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an alignment of the amino acid sequence of ArabidopsisIND1 with the Brassica napus amino acid sequences of Bn IND1 and BnIND2.

FIG. 2 depicts an alignment of the nucleotide sequence of ArabidopsisIND1 with the Brassica napus amino acid sequences of Bn IND1 and BnIND2.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides polynucleotides encoding IND sequences,transgenic plants comprising the polynucleotides and methods fromodulating lignification and/or seed dehiscence in plants. The sequencesof the invention are based, in part, on the discovery of two Brassicanapus polynucleotides that encode orthologs of the ARbidopsis IND1 geneand which can complement Arabidopsis ind1 mutants.

Generally, the nomenclature and the laboratory procedures in recombinantDNA technology described below are those well known and commonlyemployed in the art. Standard techniques are used for cloning, DNA andRNA isolation, amplification and purification. Generally enzymaticreactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like are performed according to the manufacturer'sspecifications. These techniques and various other techniques aregenerally performed according to Sambrook et al., Molecular Cloning—ALaboratory Manual, 2nd. ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., (1989) and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 2004).

II. Isolation of Nucleic Acids of the Invention

The isolation of polynucleotides of the invention may be accomplished bya number of techniques. For instance, oligonucleotide probes based onthe sequences disclosed here can be used to identify the desiredpolynucloetide in a cDNA or genomic DNA library from a desired plantspecies. To construct genomic libraries, large segments of genomic DNAare generated by random fragmentation, e.g. using restrictionendonucleases, and are ligated with vector DNA to form concatemers thatcan be packaged into the appropriate vector. Alternatively, cDNAlibraries from plants or plant parts (e.g., flowers) may be constructed.

The cDNA or genomic library can then be screened using a probe basedupon the sequence of a cloned IND1 gene such as the polynucleotidesdisclosed here. Probes may be used to hybridize with genomic DNA or cDNAsequences to isolate homologous genes in the same or different plantspecies.

Alternatively, the nucleic acids of interest can be amplified fromnucleic acid samples using amplification techniques. For instance,polymerase chain reaction (PCR) technology to amplify the sequences ofthe genes directly from mRNA, from cDNA, from genomic libraries or cDNAlibraries. PCR and other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of the desired mRNA in samples, for nucleic acidsequencing, or for other purposes.

Appropriate primers and probes for identifying genes such as IND1 fromplant tissues are generated from comparisons of the sequences providedherein. For a general overview of PCR see PCR Protocols: A Guide toMethods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White,T., eds.), Academic Press, San Diego (1990). Appropriate primers foramplification of the genomic region of Arabidopsis IND1 or the IND1 cDNAinclude the following primer pairs: 5′-gatgaaaatggaaaatggtatgtata-3′ and5′-gttcatcagggttgggagttgtg-3′. The amplification conditions aretypically as follows. Reaction components: 10 mM Tris-HCl, pH 8.3, 50 mMpotassium chloride, 1.5 mM magnesium chloride, 0.001% gelatin, 200 μMdATP, 200 μM dCTP, 200 μM dGTP, 200 μM dTTP, 0.4 μM primers, and 100units per ml Taq polymerase. Program: 96 C for 3 min., 30 cycles of 96 Cfor 45 sec., 50 C for 60 sec., 72 for 60 sec, followed by 72 C for 5min.

Polynucleotides may also be synthesized by well-known techniques asdescribed in the technical literature. See, e.g., Carruthers et al.,Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams etal., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments maythen be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

The genus of IND1 nucleic acid sequences of the invention includes genesand gene products identified and characterized by analysis using nucleicacid sequences of the invention, including SEQ ID NO:1, 7, 8 11, and 12and protein sequences of the invention, including SEQ ID NO:2, 9 and 10.IND1 sequences of the invention include nucleic acid sequences havingsubstantial identity to SEQ ID NO:1, 7, 8 11, and/or 12. IND1 sequencesof the invention also include polypeptide sequences having substantialidentity to SEQ ID NO: 2, 9 and/or 10.

III. Use of Nucleic Acids of the Invention

A. Use of Nucleic Acids of the Invention to Inhibit or Suppress GeneExpression

The invention provides methods of modulating fruit dehiscence in a plantby introducing into a plant a recombinant expression cassette comprisinga regulatory element operably linked to a IND1 polynucleotide. Theinvention also provides methods for delaying seed dispersal in a plantby suppressing expression of a nucleic acid molecule encoding an IND1gene product. In a transgenic plant of the invention, a nucleic acidmolecule, or antisense constructs thereof, encoding an IND1 gene productcan be operatively linked to an exogenous regulatory element. Theinvention provides, for example, a transgenic plant characterized bydelayed seed dispersal having an expressed nucleic acid moleculeencoding an IND1 gene product, or antisense construct thereof, that isoperatively linked to an exogenous constitutive regulatory element. Inone embodiment, the invention provides a transgenic plant that ischaracterized by delayed seed dispersal and/or modulated lignificationdue to suppression of a nucleic acid molecule encoding an IND1polypeptide. In some embodiments, suppression of IND1 expression resultsin reduced lignification in cells adjacent to the dehiscence zone (e.g.,valve margin cells, see, e.g., U.S. application Ser. No. 09/339,998,filed on Jun. 25, 1999), whereas ectopic expression results in increasedlignification.

The IND1 sequences of the invention can be used to prepare expressioncassettes useful in a number of techniques, including inhibiting,suppressing or increasing, expression or for ectopic expression. Anumber of methods can be used to inhibit gene expression in plants. Forinstance, antisense technology can be conveniently used. To accomplishthis, a nucleic acid segment from the desired gene is cloned andoperably linked to a promoter such that the antisense strand of RNA willbe transcribed. The expression cassette is then transformed into plantsand the antisense strand of RNA is produced. In plant cells, it has beensuggested that antisense RNA inhibits gene expression by preventing theaccumulation of mRNA which encodes the enzyme of interest, see, e.g.,Sheehy et al., Proc. Nat. Acad. Sci. USA, 85:8805-8809 (1988); Pnueli etal., The Plant Cell 6:175-186 (1994); and Hiatt et al., U.S. Pat. No.4,801,340.

The antisense nucleic acid sequence transformed into plants will besubstantially identical to at least a portion of the endogenous gene orgenes to be repressed. The sequence, however, does not have to beperfectly identical to inhibit expression. Thus, an antisense or sensenucleic acid molecule encoding only a portion of IND can be useful forproducing a plant in which IND1 expression is suppressed. The vectors ofthe present invention can be designed such that the inhibitory effectapplies to other proteins within a family of genes exhibiting homologyor substantial homology to the target gene.

For antisense suppression, the introduced sequence also need not be fulllength relative to either the primary transcription product or fullyprocessed mRNA. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Furthermore, the introduced sequence neednot have the same intron or exon pattern, and homology of non-codingsegments may be equally effective. Normally, a sequence of between about30 or 40 nucleotides and about full length nucleotides should be used,though a sequence of at least about 100 nucleotides is preferred, asequence of at least about 200 nucleotides is more preferred, and asequence of at least about 500 nucleotides is especially preferred.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of IND1 genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified. One class ofribozymes is derived from a number of small circular RNAs that arecapable of self-cleavage and replication in plants. The RNAs replicateeither alone (viroid RNAs) or with a helper virus (satellite RNAs).Examples include RNAs from avocado sunblotch viroid and the satelliteRNAs from tobacco ringspot virus, lucerne transient streak virus, velvettobacco mottle virus, solanum nodiflorum mottle virus and subterraneanclover mottle virus. The design and use of target RNA-specific ribozymesis described in Haseloff et al. Nature, 334:585-591 (1988).

Another method of suppression is sense suppression (also known asco-suppression). Introduction of expression cassettes in which a nucleicacid is configured in the sense orientation with respect to the promoterhas been shown to be an effective means by which to block thetranscription of target genes. For an example of the use of this methodto modulate expression of endogenous genes see, Napoli et al., The PlantCell 2:279-289 (1990); Flavell, Proc. Natl. Acad. Sci., USA 91:3490-3496(1994); Kooter and Mol, Current Opin. Biol. 4:166-171 (1993); and U.S.Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.

Generally, where inhibition of expression is desired, some transcriptionof the introduced sequence occurs. The effect may occur where theintroduced sequence contains no coding sequence per se, but only intronor untranslated sequences homologous to sequences present in the primarytranscript of the endogenous sequence. The introduced sequence generallywill be substantially identical to the endogenous sequence intended tobe repressed. This minimal identity will typically be greater than about65%, but a higher identity might exert a more effective repression ofexpression of the endogenous sequences. Substantially greater identityof more than about 80% is preferred, though about 95% to absoluteidentity would be most preferred. As with antisense regulation, theeffect should apply to any other proteins within a similar family ofgenes exhibiting homology or substantial homology.

For sense suppression, the introduced sequence in the expressioncassette, needing less than absolute identity, also need not be fulllength, relative to either the primary transcription product or fullyprocessed mRNA. This may be preferred to avoid concurrent production ofsome plants that are overexpressers. A higher identity in a shorter thanfull length sequence compensates for a longer, less identical sequence.Furthermore, the introduced sequence need not have the same intron orexon pattern, and identity of non-coding segments will be equallyeffective. Normally, a sequence of the size ranges noted above forantisense regulation is used.

Endogenous gene expression may also be suppressed by means of RNAinterference (RNAi), which uses a double-stranded RNA having a sequenceidentical or similar to the sequence of the target gene. RNAi is thephenomenon in which when a double-stranded RNA having a sequenceidentical or similar to that of the target gene is introduced into acell, the expressions of both the inserted exogenous gene and targetendogenous gene are suppressed. The double-stranded RNA may be formedfrom two separate complementry RNAs or may be a single RNA withinternally complementary sequences that form a double-stranded RNA.Although details of the mechanism of RNAi are still unknown, it isconsidered that the introduced double-stranded RNA is initially cleavedinto small fragments, which then serve as indexes of the target gene insome manner, thereby degrading the target gene. RNAi is known to be alsoeffective in plants (see, e.g., Chuang, C. F. & Meyerowitz, E. M., Proc.Natl. Acad. Sci. USA 97: 4985 (2000); Waterhouse et al., Proc. Natl.Acad. Sci. USA 95:13959-13964 (1998); Tabara et al. Science 282:430-431(1998)). For example, to achieve suppression of the expression of a DNAencoding a protein using RNAi, a double-stranded RNA having the sequenceof a DNA encoding the protein, or a substantially similar sequencethereof (including those engineered not to translate the protein) orfragment thereof, is introduced into a plant of interest. The resultingplants may then be screened for a phenotype associated with the targetprotein and/or by monitoring steady-state RNA levels for transcriptsencoding the protein. Although the genes used for RNAi need not becompletely identical to the target gene, they may be at least 70%, 80%,90%, 95% or more identical to the target gene sequence. See, e.g., U.S.Patent Publication No. 2004/0029283. The constructs encoding an RNAmolecule with a stem-loop structure that is unrelated to the target geneand that is positioned distally to a sequence specific for the gene ofinterest may also be used to inhibit target gene expression. See, e.g.,U.S. Patent Publication No. 2003/0221211.

The RNAi polynucleotides may encompass the full-length target RNA or maycorrespond to a fragment of the target RNA. In some cases, the fragmentwill have fewer than 100, 200, 300, 400, 500 600, 700, 800, 900 or 1,000nucleotides corresponding to the target sequence. In addition, in someembodiments, these fragments are at least, e.g., 50, 100, 150, 200, ormore nucleotides in length. In some cases, fragments for use in RNAiwill be at least substantially similar to regions of a target proteinthat do not occur in other proteins in the organism or may be selectedto have as little similarity to other organism transcripts as possible,e.g., selected by comparison to sequences in analyzingpublicly-available sequence databases. Thus, RNAi fragments may beselected for similarity or identity with the N terminal region of theIND1 and BIND1 sequences of the invention (i.e., those sequences lackingsignificant homology to sequences in the databases) or may be selectedfor identity or similarity to coding sequences for the bHLH domain or atleast sequences distinguishing the IND1 bHLH domain from other bHLHdomain proteins.

Expression vectors that continually express siRNA in transiently- andstably-transfected have been engineered to express small hairpin RNAs,which get processed in vivo into siRNAs molecules capable of carryingout gene-specific silencing (Brummelkamp et al., Science 296:550-553(2002), and Paddison, et al., Genes & Dev. 16:948-958 (2002)).Post-transcriptional gene silencing by double-stranded RNA is discussedin further detail by Hammond et al. Nature Rev Gen 2: 110-119 (2001),Fire et al. Nature 391: 806-811 (1998) and Timmons and Fire Nature 395:854 (1998).

One of skill in the art will recognize that using technology based onspecific nucleotide sequences (e.g., antisense or sense suppressiontechnology), families of homologous genes can be suppressed with asingle sense or antisense transcript. For instance, if a sense orantisense transcript is designed to have a sequence that is conservedamong a family of genes, then multiple members of a gene family can besuppressed. Conversely, if the goal is to only suppress one member of ahomologous gene family, then the sense or antisense transcript should betargeted to sequences with the most vairance between family members.

Another means of inhibiting IND1 function in a plant is by creation ofdominant negative mutations. In this approach, non-functional, mutantIND1 polypeptides, which retain the ability to interact with wild-typesubunits are introduced into a plant. A dominant negative construct alsocan be used to suppress IND1 expression in a plant. A dominant negativeconstruct useful in the invention generally contains a portion of thecomplete IND1 coding sequence sufficient, for example, for DNA-bindingor for a protein-protein interaction such as a homodimeric orheterodimeric protein-protein interaction but lacking thetranscriptional activity of the wild type protein. For example, acarboxy-terminal deletion mutant of AGAMOUS was used as a dominantnegative construct to suppress expression of the MADS box gene AGAMOUS(Mizukami et al., Plant Cell 8:831-844 (1996)). One skilled in the artunderstands that, similarly, a dominant negative IND1 construct can beused to suppress IND1 expression in a plant.

B. Use of Nucleic Acids of the Invention to Enhance Gene Expression

Isolated sequences prepared as described herein can also be used toprepare expression cassettes that enhance or increase endogenous IND1gene expression. Where overexpression of a gene is desired, the desiredgene from a different species may be used to decrease potential sensesuppression effects. Enhanced expression of IND1 polynucleotides isuseful, for example, to produce plants with small fruit.

Any of a number of means well known in the art can be used to increaseIND1 activity in plants. Any organ can be targeted, such as shootvegetative organs/structures (e.g. leaves, stems and tubers), roots,flowers and floral organs/structures (e.g. bracts, sepals, petals,stamens, carpels, anthers and ovules), seed (including embryo,endosperm, and seed coat) and fruit. Alternatively, one or several IND1genes can be expressed constitutively (e.g., using the CaMV 35Spromoter).

One of skill will recognize that the polypeptides encoded by the genesof the invention, like other proteins, have different domains whichperform different functions. Thus, the gene sequences need not be fulllength, so long as the desired functional domain of the protein isexpressed. As explained above, IND1 polypeptides carry a bHLH domain,which is capable of binding DNA. Thus, without being bound to anyparticular theory or mechanism, IND1 is likely to act as atranscriptional modulator.

C. Modification of Endogenous IND1 Genes

Methods for introducing genetic mutations into plant genes and selectingplants with desired traits are well known. For instance, seeds or otherplant material can be treated with a mutagenic chemical substance,according to standard techniques. Such chemical substances include, butare not limited to, the following: diethyl sulfate, ethylene imine,ethyl methanesulfonate and N-nitroso-N-ethylurea. Alternatively,ionizing radiation from sources such as, X-rays or gamma rays can beused.

Modified protein chains can also be readily designed utilizing variousrecombinant DNA techniques well known to those skilled in the art anddescribed for instance, in Sambrook et al., supra. Hydroxylamine canalso be used to introduce single base mutations into the coding regionof the gene (Sikorski, et al., (1991). Meth. Enzymol. 194: 302-318). Forexample, the chains can vary from the naturally occurring sequence atthe primary structure level by amino acid substitutions, additions,deletions, and the like. These modifications can be used in a number ofcombinations to produce the final modified protein chain.

Alternatively, homologous recombination can be used to induce targetedgene modifications by specifically targeting the IND1 gene in vivo (see,generally, Grewal and Klar, Genetics 146: 1221-1238 (1997) and Xu etal., Genes Dev. 10: 2411-2422 (1996)). Homologous recombination has beendemonstrated in plants (Puchta et al., Experientia 50: 277-284 (1994),Swoboda et al., EMBO J. 13: 484-489 (1994); Offringa et al., Proc. Natl.Acad. Sci. USA 90: 7346-7350 (1993); and Kempin et al. Nature389:802-803 (1997)).

In applying homologous recombination technology to the genes of theinvention, mutations in selected portions of an IND1 gene sequences(including 5′ upstream, 3′ downstream, and intragenic regions) such asthose disclosed here are made in vitro and then introduced into thedesired plant using standard techniques. Since the efficiency ofhomologous recombination is known to be dependent on the vectors used,use of dicistronic gene targeting vectors as described by Mountford etal., Proc. Natl. Acad. Sci. USA 91: 4303-4307 (1994); and Vaulont etal., Transgenic Res. 4: 247-255 (1995) are conveniently used to increasethe efficiency of selecting for altered IND1 gene expression intransgenic plants. The mutated gene will interact with the targetwild-type gene in such a way that homologous recombination and targetedreplacement of the wild-type gene will occur in transgenic plant cells,resulting in suppression of IND1 activity.

Alternatively, oligonucleotides composed of a contiguous stretch of RNAand DNA residues in a duplex conformation with double hairpin caps onthe ends can be used. The RNA/DNA sequence is designed to align with thesequence of the target IND1 gene and to contain the desired nucleotidechange. Introduction of the chimeric oligonucleotide on anextrachromosomal T-DNA plasmid results in efficient and specific IND1gene conversion directed by chimeric molecules in a small number oftransformed plant cells. This method is described in Cole-Strauss etal., Science 273:1386-1389 (1996) and Yoon et al., Proc. Natl. Acad.Sci. USA 93: 2071-2076 (1996).

In other embodiments, the promoters derived from the IND1 genes of theinvention can be used to drive expression of heterologous genes in anvalve margin-specific manner. Suitable structural genes that could beused for this purpose include genes encoding cytotoxic proteins asdiscussed below.

Typically, desired promoters are identified by analyzing the 5′sequences of a genomic clone corresponding to the IND1 genes describedhere. Sequences characteristic of promoter sequences can be used toidentify the promoter. Sequences controlling eukaryotic gene expressionhave been extensively studied. For instance, promoter sequence elementsinclude the TATA box consensus sequence (TATAAT), which is usually 20 to30 base pairs upstream of the transcription start site. In mostinstances the TATA box is required for accurate transcriptioninitiation. In plants, further upstream from the TATA box, at positions−80 to −100, there is typically a promoter element with a series ofadenines surrounding the trinucleotide G (or T) N G. J. Messing et al.,in GENETIC ENGINEERING IN PLANTS, pp. 221-227 (Kosage, Meredith andHollaender, eds. (1983)).

A number of methods are known to those of skill in the art foridentifying and characterizing promoter regions in plant genomic DNA(see, e.g., Jordano, et al., Plant Cell, 1: 855-866 (1989); Bustos, etal., Plant Cell, 1:839-854 (1989); Green, et al., EMBO J. 7, 4035-4044(1988); Meier, et al., Plant Cell, 3, 309-316 (1991); and Zhang, et al.,Plant Physiology 110: 1069-1079 (1996)).

IV. Preparation of Recombinant Vectors

To use isolated sequences in the above techniques, recombinant DNAvectors suitable for transformation of plant cells are prepared.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, for example, Weising et al. Ann. Rev. Genet. 22:421-477 (1988). ADNA sequence coding for the desired polypeptide, for example a cDNAsequence encoding a full length protein, will preferably be combinedwith transcriptional and translational initiation regulatory sequenceswhich will direct the transcription of the sequence from the gene in theintended tissues of the transformed plant.

For example, for overexpression, a plant promoter fragment may beemployed which will direct expression of the gene in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumafaciens, and other transcription initiationregions from various plant genes known to those of skill.

Alternatively, the plant promoter may direct expression of thepolynucleotide of the invention in a specific tissue (tissue-specificpromoters) or may be otherwise under more precise environmental control(inducible promoters). Examples of tissue-specific promoters underdevelopmental control include promoters that initiate transcription onlyin certain tissues, such as fruit, seeds, or flowers. As noted above,the promoters from the IND1 genes described here are particularly usefulfor directing gene expression so that a desired gene product is locatedin the valve margin of fruit. Other suitable promoters include thosefrom genes such as SHP1 or SHP2 (Savidge, B., Rounsley, S. D., andYanofsky, M. F. (1995) Plant Cell 7: 721-733). Examples of environmentalconditions that may affect transcription by inducible promoters includeanaerobic conditions, elevated temperature, or the presence of light.

If proper polypeptide expression is desired, a polyadenylation region atthe 3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions)from genes of the invention will typically comprise a marker gene thatconfers a selectable phenotype on plant cells. For example, the markermay encode biocide resistance, particularly antibiotic resistance, suchas resistance to kanamycin, G418, bleomycin, hygromycin, or herbicideresistance, such as resistance to chlorosluforon or Basta.

IND1 nucleic acid sequences of the invention are expressed recombinantlyin plant cells to enhance and increase levels of endogenous IND1polypeptides. Alternatively, antisense or other IND1 constructs(described above) are used to suppress IND1 levels of expression. Avariety of different expression constructs, such as expression cassettesand vectors suitable for transformation of plant cells can be prepared.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, e.g., Weising et al. Ann. Rev. Genet. 22:421-477 (1988). A DNAsequence coding for a IND1 polypeptide, e.g., a cDNA sequence encoding afull length protein, can be combined with cis-acting (promoter) andtrans-acting (enhancer) transcriptional regulatory sequences to directthe timing, tissue type and levels of transcription in the intendedtissues of the transformed plant. Translational control elements canalso be used.

The invention provides an IND1 nucleic acid operably linked to apromoter which, in a preferred embodiment, is capable of driving thetranscription of the IND1 coding sequence in plants. The promoter canbe, e.g., derived from plant or viral sources. The promoter can be,e.g., constitutively active, inducible, or tissue specific. Inconstruction of recombinant expression cassettes, vectors, transgenics,of the invention, a different promoters can be chosen and employed todifferentially direct gene expression, e.g., in some or all tissues of aplant or animal. Typically, as discussed above, desired promoters areidentified by analyzing the 5′ sequences of a genomic clonecorresponding to the IND1 genes described here.

A. Constitutive Promoters

A promoter fragment can be employed which will direct expression of IND1nucleic acid in all transformed cells or tissues, e.g. as those of aregenerated plant. The term “constitutive regulatory element” means aregulatory element that confers a level of expression upon anoperatively linked nucleic molecule that is relatively independent ofthe cell or tissue type in which the constitutive regulatory element isexpressed. A constitutive regulatory element that is expressed in aplant generally is widely expressed in a large number of cell and tissuetypes. Promoters that drive expression continuously under physiologicalconditions are referred to as “constitutive” promoters and are activeunder most environmental conditions and states of development or celldifferentiation.

A variety of constitutive regulatory elements useful for ectopicexpression in a transgenic plant are well known in the art. Thecauliflower mosaic virus 35S (CaMV 35S) promoter, for example, is awell-characterized constitutive regulatory element that produces a highlevel of expression in all plant tissues (Odell et al., Nature313:810-812 (1985)). The CaMV 35S promoter can be particularly usefuldue to its activity in numerous diverse plant species (Benfey and Chua,Science 250:959-966 (1990); Futterer et al., Physiol. Plant 79:154(1990); Odell et al., supra, 1985). A tandem 35S promoter, in which theintrinsic promoter element has been duplicated, confers higherexpression levels in comparison to the unmodified 35S promoter (Kay etal., Science 236:1299 (1987)). Other useful constitutive regulatoryelements include, for example, the cauliflower mosaic virus 19Spromoter; the Figwort mosaic virus promoter; and the nopaline synthase(nos) gene promoter (Singer et al., Plant Mol. Biol. 14:433 (1990); An,Plant Physiol. 81:86 (1986)).

Additional constitutive regulatory elements including those forefficient expression in monocots also are known in the art, for example,the pEmu promoter and promoters based on the rice Actin-1 5′ region(Last et al., Theor. Appl. Genet. 81:581 (1991); Mcelroy et al., Mol.Gen. Genet. 231:150 (1991); Mcelroy et al., Plant Cell 2:163 (1990)).Chimeric regulatory elements, which combine elements from differentgenes, also can be useful for ectopically expressing a nucleic acidmolecule encoding an IND1 polynucleotide (Comai et al., Plant Mol. Biol.15:373 (1990)).

Other examples of constitutive promoters include the 1′- or 2′-promoterderived from T-DNA of Agrobacterium tumafaciens (see, e.g., Mengiste(1997) supra; O'Grady (1995) Plant Mol. Biol. 29:99-108); actinpromoters, such as the Arabidopsis actin gene promoter (see, e.g., Huang(1997) Plant Mol. Biol. 1997 33:125-139); alcohol dehydrogenase (Adh)gene promoters (see, e.g., Millar (1996) Plant Mol. Biol. 31:897-904);ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139(1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al., Mol.Gen. Genet. 251:196-203 (1996)), the gene encoding stearoyl-acyl carrierprotein desaturase from Brassica napus (Genbank No. X74782, Solocombe etal. Plant Physiol. 104:1167-1176 (1994)), GPc1 from maize (GenBank No.X15596, Martinez et al. J. Mol. Biol. 208:551-565 (1989)), Gpc2 frommaize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol. 33:97-112(1997)), other transcription initiation regions from various plant genesknown to those of skill. See also Holtorf Plant Mol. Biol. 29:637-646(1995).

B. Inducible Promoters

Alternatively, a plant promoter may direct expression of the IND1nucleic acid of the invention under the influence of changingenvironmental conditions or developmental conditions. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions, elevated temperature, drought,or the presence of light. Such promoters are referred to herein as“inducible” promoters. For example, the invention incorporates thedrought-inducible promoter of maize (Busk (1997) supra); the cold,drought, and high salt inducible promoter from potato (Kirch (1997)Plant Mol. Biol. 33:897-909).

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

Plant promoters which are inducible upon exposure to chemicals reagentswhich can be applied to the plant, such as herbicides or antibiotics,are also used to express the nucleic acids of the invention. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568-577); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. IND1 coding sequence can alsobe under the control of, e.g., a tetracycline-inducible promoter, e.g.,as described with transgenic tobacco plants containing the Avena sativaL. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J.11:465-473); or, a salicylic acid-responsive element (Stange (1997)Plant J. 11:1315-1324; Uknes et al., Plant Cell 5:159-169 (1993); Bi etal., Plant J. 8:235-245 (1995)).

Particularly useful inducible regulatory elements includecopper-inducible regulatory elements (Mett et al., Proc. Natl. Acad.Sci. USA 90:4567-4571 (1993); Furst et al., Cell 55:705-717 (1988));tetracycline and chlor-tetracycline-inducible regulatory elements (Gatzet al., Plant J. 2:397-404 (1992); Roder et al., Mol. Gen. Genet.243:32-38 (1994); Gatz, Meth. Cell Biol. 50:411-424 (1995)); ecdysoneinducible regulatory elements (Christopherson et al., Proc. Natl. Acad.Sci. USA 89:6314-6318 (1992); Kreutzweiser et al., Ecotoxicol. Environ.Safety 28:14-24 (1994)); heat shock inducible regulatory elements(Takahashi et al., Plant Physiol. 99:383-390 (1992); Yabe et al., PlantCell Physiol. 35:1207-1219 (1994); Ueda et al., Mol. Gen. Genet.250:533-539 (1996)); and lac operon elements, which are used incombination with a constitutively expressed lac repressor to confer, forexample, IPTG-inducible expression (Wilde et al., EMBO J. 11:1251-1259(1992)). An inducible regulatory element useful in the transgenic plantsof the invention also can be, for example, a nitrate-inducible promoterderived from the spinach nitrite reductase gene (Back et al., Plant Mol.Biol. 17:9 (1991)) or a light-inducible promoter, such as thatassociated with the small subunit of RuBP carboxylase or the LHCP genefamilies (Feinbaum et al., Mol. Gen. Genet. 226:449 (1991); Lam andChua, Science 248:471 (1990)).

C. Tissue-Specific Promoters

Alternatively, the plant promoter may direct expression of thepolynucleotide of the invention in a specific tissue (tissue-specificpromoters). Tissue specific promoters are transcriptional controlelements that are only active in particular cells or tissues at specifictimes during plant development, such as in vegetative tissues orreproductive tissues. Promoters from the IND1 genes of the invention areparticularly useful for tissue-specific direction of gene expression sothat a desired gene product is generated only or preferentially inembryos or seeds, as described below.

Examples of tissue-specific promoters under developmental controlinclude promoters that initiate transcription only (or primarily only)in certain tissues, such as vegetative tissues, e.g., roots or leaves,or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols,flowers, or any embryonic tissue. Reproductive tissue-specific promotersmay be, e.g., ovule-specific, embryo-specific, endosperm-specific,integument-specific, seed and seed coat-specific, pollen-specific,petal-specific, sepal-specific, or some combination thereof.

The invention provides a transgenic plant that is characterized bydelayed seed dispersal due to expression of a nucleic acid moleculeencoding an IND1 gene product, or an antisense construct thereof,operatively linked to a dehiscence zone-selective regulatory element.The dehiscence zone-selective regulatory element can be, for example, anSHP1 regulatory element or SHP2 regulatory element. The SHP1 regulatoryelement can be derived from the Arabidopsis SHP1 genomic sequencedisclosed herein as SEQ ID NO:5 and can be, for example, a 5′ regulatorysequence or intronic regulatory element. Similarly, the SHP2 regulatoryelement can be derived from the Arabidopsis SHP2 genomic sequencedisclosed herein as SEQ ID NO:6 and can be, for example, a 5′ regulatorysequence or intronic regulatory element.

A dehiscence zone-selective regulatory element can be derived from agene that is an ortholog of Arabidopsis IND1 and is selectivelyexpressed in the valve margin or dehiscence zone of a seed plant. Adehiscence zone-selective regulatory element can be derived, forexample, from an IND1 ortholog of the Brassicaceae, such as a Brassicanapus, Brassica oleracea, Brassica campestris, Brassica juncea, Brassicanigra or Brassica carinata IND1 ortholog. A dehiscence zone-selectiveregulatory element can be derived, for example, from an IND1 canolaortholog. A dehiscence zone-selective regulatory element also can bederived, for example, from a leguminous IND1 ortholog, such as asoybean, pea, chickpea, moth bean, broad bean, kidney bean, lima bean,lentil, cowpea, dry bean, peanut, alfalfa, lucerne, birdsfoot trefoil,clover, stylosanthes, lotononis bainessii, or sainfoin IND1 ortholog.

Dehiscence zone-selective regulatory elements also can be derived from avariety of other genes that are selectively expressed in the valvemargin or dehiscence zone of a seed plant. For example, the rapeseedgene RDPG1 is selectively expressed in the dehiscence zone (Petersen etal., Plant Mol. Biol. 31:517-527 (1996)). Thus, the RDPG1 promoter or anactive fragment thereof can be a dehiscence zone-selective regulatoryelement as defined herein. Additional genes such as the rapeseed geneSAC51 also are known to be selectively expressed in the dehiscence zone;the SAC51 promoter or an active fragment thereof also can be adehiscence zone-selective regulatory element of the invention (Coupe etal., Plant Mol. Biol. 23:1223-1232 (1993)). The skilled artisanunderstands that a regulatory element of any such gene selectivelyexpressed in cells of the valve margin or dehiscence zone can be adehiscence zone-selective regulatory element as defined herein.

Additional dehiscence zone-selective regulatory elements can beidentified and isolated using routine methodology. Differentialscreening strategies using, for example, RNA prepared from thedehiscence zone and RNA prepared from adjacent pod material can be usedto isolate cDNAs selectively expressed in cells of the dehiscence zone(Coupe et al., supra, 1993); subsequently, the corresponding genes areisolated using the cDNA sequence as a probe.

Enhancer trap or gene trap strategies also can be used to identify andisolate a dehiscence zone-selective regulatory element of the invention(Sundaresan et al., supra, 1995; Koncz et al., Proc. Natl. Acad. Sci.USA 86:8467-8471 (1989); Kertbundit et al., Proc. Natl. Acad. Sci. USA88:5212-5216 (1991); Topping et al., Development 112:1009-1019 (1991)).Enhancer trap elements include a reporter gene such as GUS with a weakor minimal promoter, while gene trap elements lack a promoter sequence,relying on transcription from a flanking chromosomal gene for reportergene expression. Transposable elements included in the constructsmediate fusions to endogenous loci; constructs selectively expressed inthe valve margin or dehiscence zone are identified by their pattern ofexpression. With the inserted element as a tag, the flanking dehiscencezone-selective regulatory element is cloned using, for example, inversepolymerase chain reaction methodology (see, for example, Aarts et al.,Nature 363:715-717 (1993); see also, Ochman et al., “Amplification ofFlanking Sequences by Inverse PCR,” in Innis et al., supra, 1990). TheAc/Ds transposition system of Sundaresan et al., Genes. Devel.9:1797-1810 (1995), can be particularly useful in identifying andisolating a dehiscence zone-selective regulatory element of theinvention.

Dehiscence zone-selective regulatory elements also can be isolated byinserting a library of random genomic DNA fragments in front of apromoterless reporter gene and screening transgenic plants transformedwith the library for dehiscence zone-selective reporter gene expression.The promoterless vector pROA97, which contains the npt gene and the GUSgene each under the control of the minimal 35S promoter, can be usefulfor such screening. The genomic library can be, for example, Sau3Afragments of Arabidopsis thaliana genomic DNA or genomic DNA from, forexample, another Brassicaceae of interest (Ott et al., Mol. Gen. Genet.223:169-179 (1990); Claes et al., The Plant Journal 1:15-26 (1991)).

Dehiscence zone-selective expression of a regulatory element of theinvention can be demonstrated or confirmed by routine techniques, forexample, using a reporter gene and in situ expression analysis. The GUSand firefly luciferase reporters are particularly useful for in situlocalization of plant gene expression (Jefferson et al., EMBO J. 6:3901(1987); Ow et al., Science 334:856 (1986)), and promoterless vectorscontaining the GUS expression cassette are commercially available, forexample, from Clontech (Palo Alto, Calif.). To identify a dehiscencezone-selective regulatory element of interest such as an IND1 regulatoryelement, one or more nucleotide portions of the IND1 gene can begenerated using enzymatic or PCR-based methodology (Glick and Thompson,supra, 1993; Innis et al., supra, 1990); the resulting segments arefused to a reporter gene such as GUS and analyzed as described above.

Other tissue-specific promoters include seed promoters. Suitableseed-specific promoters are derived from the following genes: MAC1 frommaize (Sheridan (1996) Genetics 142:1009-1020); Cat3 from maize (GenBankNo. L05934, Abler (1993) Plant Mol. Biol. 22:10131-1038); vivparous-1from Arabidopsis (Genbank No. U93215); atmyc1 from Arabidopsis (Urao(1996) Plant Mol. Biol. 32:571-57; Conceicao (1994) Plant 5:493-505);napA from Brassica napus (GenBank No. J02798, Josefsson (1987) JBL26:12196-1301); and the napin gene family from Brassica napus (Sjodahl(1995) Planta 197:264-271).

A variety of promoters specifically active in vegetative tissues, suchas leaves, stems, roots and tubers, can also be used to express the IND1nucleic acids of the invention. For example, promoters controllingpatatin, the major storage protein of the potato tuber, can be used,see, e.g., Kim (1994) Plant Mol. Biol. 26:603-615; Martin (1997) PlantJ. 11:53-62. The ORF13 promoter from Agrobacterium rhizogenes whichexhibits high activity in roots can also be used (Hansen (1997) Mol.Gen. Genet. 254:337-343. Other useful vegetative tissue-specificpromoters include: the tarin promoter of the gene encoding a globulinfrom a major taro (Colocasia esculenta L. Schott) corm protein family,tarin (Bezerra (1995) Plant Mol. Biol. 28:137-144); the curculinpromoter active during taro corm development (de Castro (1992) PlantCell 4:1549-1559) and the promoter for the tobacco root-specific geneTobRB7, whose expression is localized to root meristem and immaturecentral cylinder regions (Yamamoto (1991) Plant Cell 3:371-382).

Leaf-specific promoters, such as the ribulose biphosphate carboxylase(RBCS) promoters can be used. For example, the tomato RBCS1, RBCS2 andRBCS3A genes are expressed in leaves and light-grown seedlings, onlyRBCS1 and RBCS2 are expressed in developing tomato fruits (Meier (1997)FEBS Lett. 415:91-95). A ribulose bisphosphate carboxylase promotersexpressed almost exclusively in mesophyll cells in leaf blades and leafsheaths at high levels, described by Matsuoka (1994) Plant J. 6:311-319,can be used. Another leaf-specific promoter is the light harvestingchlorophyll a/b binding protein gene promoter, see, e.g., Shiina (1997)Plant Physiol. 115:477-483; Casal (1998) Plant Physiol. 116:1533-1538.The Arabidopsis thaliana myb-related gene promoter (Atmyb5) described byLi (1996) FEBS Lett. 379:117-121, is leaf-specific. The Atmyb5 promoteris expressed in developing leaf trichomes, stipules, and epidermal cellson the margins of young rosette and cauline leaves, and in immatureseeds. Atmyb5 mRNA appears between fertilization and the 16 cell stageof embryo development and persists beyond the heart stage. A leafpromoter identified in maize by Busk (1997) Plant J. 11:1285-1295, canalso be used.

Another class of useful vegetative tissue-specific promoters aremeristematic (root tip and shoot apex) promoters. For example, the“SHOOTMERISTEMLESS” and “SCARECROW” promoters, which are active in thedeveloping shoot or root apical meristems, described by Di Laurenzio(1996) Cell 86:423-433; and, Long (1996) Nature 379:66-69; can be used.Another useful promoter is that which controls the expression of3-hydroxy-3-methylglutaryl coenzyme A reductase HMG2 gene, whoseexpression is restricted to meristematic and floral (secretory zone ofthe stigma, mature pollen grains, gynoecium vascular tissue, andfertilized ovules) tissues (see, e.g., Enjuto (1995) Plant Cell.7:517-527). Also useful are kn1-related genes from maize and otherspecies which show meristem-specific expression, see, e.g., Granger(1996) Plant Mol. Biol. 31:373-378; Kerstetter (1994) Plant Cell6:1877-1887; Hake (1995) Philos. Trans. R. Soc. Lond. B. Biol. Sci.350:45-51. For example, the Arabidopsis thaliana KNAT1 promoter. In theshoot apex, KNAT1 transcript is localized primarily to the shoot apicalmeristem; the expression of KNAT1 in the shoot meristem decreases duringthe floral transition and is restricted to the cortex of theinflorescence stem (see, e.g., Lincoln (1994) Plant Cell 6:1859-1876).

One of skill will recognize that a tissue-specific promoter may driveexpression of operably linked sequences in tissues other than the targettissue. Thus, as used herein a tissue-specific promoter is one thatdrives expression preferentially in the target tissue, but may also leadto some expression in other tissues as well.

In another embodiment, a IND1 nucleic acid is expressed through atransposable element. This allows for constitutive, yet periodic andinfrequent expression of the constitutively active polypeptide. Theinvention also provides for use of tissue-specific promoters derivedfrom viruses which can include, e.g., the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassaya vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

V. Production of Transgenic Plants

DNA constructs of the invention may be introduced into the genome of thedesired plant host by a variety of conventional techniques. For example,the DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation and microinjectionof plant cell protoplasts, or the DNA constructs can be introduceddirectly to plant tissue using ballistic methods, such as DNA particlebombardment. Alternatively, the DNA constructs may be combined withsuitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct and adjacent marker into the plant cell DNA when the cell isinfected by the bacteria.

Microinjection techniques are known in the art and well described in thescientific and patent literature. The introduction of DNA constructsusing polyethylene glycol precipitation is described in Paszkowski etal. EMBO J. 3:2717-2722 (1984). Electroporation techniques are describedin Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistictransformation techniques are described in Klein et al. Nature 327:70-73(1987).

Agrobacterium tumefaciens-mediated transformation techniques, includingdisarming and use of binary vectors, are well described in thescientific literature. See, for example Horsch et al. Science233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803(1983).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotypesuch as seedlessness. Such regeneration techniques rely on manipulationof certain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker which has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee etal. Ann. Rev. of Plant Phys. 38:467-486 (1987).

The nucleic acids of the invention can be used to confer desired traitson essentially any plant. Thus, the invention has use over a broad rangeof plants, including species from the genera Asparagus, Atropa, Avena,Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus,Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum,Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot,Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea,Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum,Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea. A useful plant ofthe invention can be a dehiscent seed plant, and a particularly usefulplant of the invention can be a member of the Brassicaceae, such asrapeseed, or a member of the Fabaceae, such as a soybean, pea, lentil orbean plant.

In one embodiment, the invention provides a dehiscent seed plant that ischaracterized by delayed seed dispersal due to suppressed expression ofa nucleic acid molecule encoding an IND1 gene product in the dehiscentseed plant. As used herein, the term “dehiscent seed plant” means aplant that produces a dry dehiscent fruit, which has fruit walls thatopen to permit escape of the seeds contained therein. Dehiscent fruitscommonly contain several seeds and include the fruits known, forexample, as legumes, capsules and siliques.

In one embodiment, the invention provides a plant that is characterizedby delayed seed dispersal due to suppressed expression of a nucleic acidmolecule encoding an IND1 gene product (e.g., substantially identical toSEQ ID NOs:2, 9, or 10), where the plant is a member of theBrassicaceae. The Brassicaceae, commonly known as the Brassicas, are adiverse group of crop plants with great economic value worldwide (see,for example, Williams and Hill, Science 232:1385-1389 (1986), which isincorporated herein by reference). The Brassicaceae produce seed oilsfor margarine, salad oil, cooking oil, plastic and industrial uses;condiment mustard; leafy, stored, processed and pickled vegetables;animal fodders and green manures for soil rejuvenation. A particularlyuseful non-naturally occurring Brassica plant of the invention is theoilseed plant canola.

There are six major Brassica species of economic importance, eachcontaining a range of plant forms. Brassica napus includes plants suchas the oilseed rapes and rutabaga. Brassica oleracea are the cole cropssuch as cabbage, cauliflower, kale, kohlrabi and Brussels sprouts.Brassica campestris (Brassica rapa) includes plants such as Chinesecabbage, turnip and pak choi. Brassica juncea includes a variety ofmustards; Brassica nigra is the black mustard; and Brassica carinata isEthiopian mustard. The skilled artisan understands that any member ofthe Brassicaceae can be modified as disclosed herein to produce anon-naturally occurring Brassica plant characterized by delayed seeddispersal.

In a second embodiment, the invention provides a plant that ischaracterized by delayed seed dispersal due to suppressed expression ofa nucleic acid molecule encoding an IND1 gene product, where the plantis a member of the Fabaceae. The Fabaceae, which are commonly known asmembers of the pea family, are plants that produce a characteristic drydehiscent fruit known as a legume. The legume is derived from a singlecarpel and dehisces along the suture of the carpel margins and along themedian vein. The Fabaceae encompass both grain legumes and foragelegumes. Grain legumes include, for example, soybean (glycine), pea,chickpea, moth bean, broad bean, kidney bean, lima bean, lentil, cowpea,dry bean and peanut. Forage legumes include alfalfa, lucerne, birdsfoottrefoil, clover, stylosanthes species, lotononis bainessii and sainfoin.The skilled artisan will recognize that any member of the Fabaceae canbe modified as disclosed herein to produce a non-naturally occurringplant of the invention characterized by delayed seed dispersal.

A non-naturally occurring plant of the invention characterized bydelayed seed dispersal also can be a member of the plant genus Cuphea(family Lythraceae). A Cuphea plant is particularly valuable sinceCuphea oilseeds contain industrially and nutritionally importantmedium-chain fatty acids, especially lauric acid, which is currentlysupplied only by coconut and palm kernel oils.

A non-naturally occurring plant of the invention also can be, forexample, one of the monocotyledonous grasses, which produce many of thevaluable small-grain cereal crops of the world. Suppression of IND1expression as described above, can be useful in generating anon-naturally occurring small grain cereal plant, such as a barley,wheat, oat, rye, orchard grass, guinea grass, sorghum or turf grassplant characterized by delayed seed dispersal.

VI. Additional Modifications that Modulate Seed Dispersal

It should be recognized that a plant of the invention, which contains anexogenous IND1 polynucleotide, also can contain one or more additionalmodifications, including naturally and non-naturally occurringmodifications, that can modulate the delay in seed dispersal. Forexample, the plant hormone ethylene promotes fruit dehiscence, andmodified expression or activity of positive or negative regulators ofthe ethylene response can be included in a plant of the invention (see,generally, Meakin and Roberts, J. Exp. Botany 41:1003-1011 (1990);Ecker, Science 268:667-675 (1995); Chao et al., Cell 89:1133-1144(1997)).

Mutations in positive regulators of the ethylene response show areduction or absence of responsiveness to treatment with exogenousethylene. Arabidopsis mutations in positive regulators of the ethyleneresponse include mutations in etr, which inactivate a histidine kinaseethylene receptor (Bleeker et al., Science 241:1086-1089 (1988);Schaller and Bleeker, Science 270:1809-1811 (1995)); ers (Hua et al.,Science 269:1712-1714 (1995)); ein2 (Guzman and Ecker, Plant Cell 2:513(1990)); ein3 (Rothenberg and Ecker, Sem. Dev. Biol. Plant Dev. Genet.4:3-13 (1993); Kieber and Ecker, Trends Genet. 9:356-362 (1993)); ain1(van der Straeten et al., Plant Physiol. 102:401-408 (1993)); eti(Harpham et al., An. Bot. 68:55 (1991)) and ein4, ein5, ein6, and ein7(Roman et al., Genetics 139: 1393-1409 (1995)). Similar geneticfunctions are found in other plant species; for example, the never-ripemutation corresponds to etr and confers ethylene insensitivity in tomato(Lanahan et al., The Plant Cell 6:521-530 (1994); Wilkinson et al.,Science 270:1807-1809 (1995)). A plant of the invention can include amodification that results in altered expression or activity of any suchpositive regulator of the ethylene response. A mutation in a positiveregulator, for example, can be included in a plant of the invention andcan modify the delay in seed dispersal in such plants, for example, byfurther postponing the delay in seed dispersal.

Mutations in negative regulators of the ethylene response displayethylene responsiveness in the absence of exogenous ethylene. Suchmutations include those relating to ethylene overproduction, forexample, the eto1, eto2, and eto3 mutants, and those relating toconstitutive activation of the ethylene signalling pathway, for example,mutations in CTR1, a negative regulator with sequence similarity to theRaf family of protein kinases (Kieber et al., Cell 72:427-441 (1993),which is incorporated herein by reference). A plant of the invention caninclude a modification that results in altered expression or activity ofany such negative regulator of the ethylene response. A mutationresulting in ethylene responsiveness in the absence of exogenousethylene, for example, can be included in a non-naturally occurringplant of the invention and can modify, for example, diminish, the delayin seed dispersal.

Fruit morphological mutations also can be included in a plant of theinvention. Such mutations include those in carpel identity genes such asAGAMOUS (Bowman et al., supra, 1989; Yanofsky et al., supra, 1990) andin genes required for normal fruit development such as ETTIN, CRABSCLAW, SPATULA, AGL8 and TOUSLED (Sessions et al., Development121:1519-1532 (1995); Alvarez and Smyth, Flowering Newsletter 23:12-17(1997); and Roe et al., Cell 75:939-950 (1993)). Thus, it is understoodthat a plant of the invention can include one or more additional geneticmodifications, which can diminish or enhance the delay in seeddispersal.

EXAMPLES

The following examples are offered to illustrate, but no to limit theclaimed invention.

Example 1

The GT140 valve margin marker (Sundaresan, V., et al. Genes Dev. 9,1797-1810 (1995)) is expressed at the valve margin of the developinggynoecium just prior to fertilization (stage 13) and this patternpersists in the mature fruit (stage 17). As expression of this marker islargely absent from the valve margins of shp1 shp2 indehiscent fruits(Liljegren, S. J., et al. Nature, 404(6779):766-70 (2000)), it wasexpected that the gene corresponding to this marker might also beinvolved in valve margin development and be required for fruitdehiscence.

To isolate flanking genomic sequence from the GT140 marker insertionsite, TAIL/PCR was performed as previously described (Tsugeki, R., etal. Plant J. 10, 479-489 (1996)). Subsequent sequencing of the isolatedPCR products demonstrated that they correspond to a fully sequenced BACfrom chromosome 4, available in the public database as part of theArabidopsis Genome Initiative. The GT140 insertion is located betweentwo genes, one encoding a predicted basic helix-loop-helix (bHLH)transcription factor and the other representing a novel gene.

Through several lines of subsequent investigation, it was confirmed thatthe bHLH transcription factor (herein referred to as IND1 as notedbelow) was the relevant gene (SEQ ID NO:1). Promoter/enhancer::GUSfusions of the IND1 gene were introduced into wild-type plants and foundto express GUS in an identical pattern to that of the GT140 marker line.Interestingly, approximately 25% of the transgenic lines failed toexpress significant GUS activity and displayed an indehiscent phenotype.The most likely explanation of these results is that the IND1::GUSfusions, as well as of the endogenous IND1 gene, were cosuppressed.Subsequent RNA blotting confirmed a down regulation of the IND1 gene inthese lines, and further RNA blotting showed, as expected, a decrease inIND1 gene expression in shp1 shp2 fruits.

In parallel to the studies of the GT140 valve margin marker describedabove, screens for Arabidopsis mutants producing indehiscent fruits werealso carried out. Besides obtaining additional alleles of SHP1 and SHP2through EMS mutagenesis of shp2-1 and shp1-1 seed stocks, indehiscentmutants that were not allelic to either SHP1 or SHP2, respectively werealso obtained. Because the GT140 studies suggested the possibility thatone or more of these indehiscent mutants might correspond to the IND1gene, IND1 from several of these mutants was cloned and sequenced. Fouralleles represent independent mutant alleles of IND1. The strongestallele, ind1-2, contains a single nucleotide deletion within codon 55that results in a frameshift and production of a truncated protein of 64rather than 198 amino acids. The ind1-1 and ind1-3 alleles containnucleotide substitutions at codons 141 and 128 that changes a leucineamino acid to a phenylalanine and an arginine to a histidine,respectively. These affected amino acids are both at conserved positionswithin the bHLH domain. The ind1-4 allele contains a nucleotidesubstitution at codon 92 that changes a glutamine to a stop codon,causing production of a truncated protein of 91 amino acids. Sinceinactivation of this bHLH transcription factor prevents fruitdehiscence, the gene is referred to as INDEHISCENT1 (IND1) and themutant as, ind1. To date, ind1 represents the only reported single genemutation in Arabidopsis that specifically blocks fruit dehiscence.

To determine whether lignified margin cells are also affected bymutations in IND, we examined the lignification pattern of ind fruitcompared to wild-type. While lignification of the vascular bundles andinner valve layer appear unaffected in ind fruit, we observed nolignified cells throughout the margins of ind-2 fruit. As marginlignification is only partially affected in shp fruit, and unaffected inalc fruit (Liljegren, S. J., et al. Nature, 404(6779):766-70 (2000),Rajani, S., and Sundaresan, V. Curr. Biol. 11:1914-1922 (2001)), theseresults indicate that IND is primarily responsible for controlling thelignification of margin cells. Interestingly, the margins of ind-1fruit, like alc fruit, are lignified, suggesting that the role of IND inseparation zone specification is genetically distinct from its role(s)in margin constriction and lignification.

IND Regulates Expression of the YJ80 Margin Marker

To further monitor the effect of mutations in IND on cellulardifferentiation at the margin, expression of molecular markers derivedfrom an enhancer trap screen (Eshed, Y., et al. Cell 99, 199-209 (1999))was examined in ind fruit compared to wild-type. We discovered that theexpression pattern of one marker, YJ80, is dramatically affected bymutations in IND. In wild-type fruit, YJ80 is expressed in stripes atthe margin, in the guard cells scattered throughout the valves, and inthe seed abscission zone. Mutations in IND completely disrupt expressionof this marker throughout the margins, whereas the other fruitexpression domains are unaffected.

Since we could detect differences between the margin defects of ind andshp1 shp2 fruit, and the phenotype of alc fruit is clearly distinct fromthat of ind and shp1 shp2, we expected that the phenotypes of thesethree mutants might be further distinguished with margin markers.Indeed, examination of the YJ80 marker in shp1 shp2 and alc fruitrevealed that this marker is still present at the apical fruit marginsof both mutants, although expression of the marker is disrupted at thebasal margins of shp1 shp2 fruit, and at the central margins of alcfruit. These results correspond with our observations that apical margindevelopment is more severely affected in ind fruit than in shp1 shp2fruit, and further suggest that IND may be the key regulator of the genecorresponding to YJ80.

IND Encodes the GT140 bHLH Transcription Factor

Through our previous studies, we identified a margin-specific marker,GT140, that is largely absent from the margins of both shp1 shp2 and35S::FUL indehiscent fruit (Sundaresan, Genes Dev. 9:1797-1810 (1995),Liljegren et al, supra, Ferrándiz, et al. Science 289, 436-438 (2000)).Since these results suggested that the gene corresponding to GT140 couldbe involved in margin development, we isolated genomic sequence flankingthe Ds transposon using TAIL/PCR (Tsugeki, Plant J. 10, 479-489 (1996)).The insertion site was found to be on chromosome 4 between two predictedORFs, At4g00120 and At4g00130. Subsequent analysis of At4g00120demonstrated that a genomic fragment containing 2.6 kb from the promoterregion directed expression of β-glucuronidase in the samemargin-specific pattern as GT140. Furthermore, approximately 25% of thetransgenic lines failed to show significant GUS activity and producedindehiscent fruit, suggesting that At4g00120 was co-suppressed in theselines and could be required for fruit dehiscence.

At4g00120, an open reading frame with no introns, encodes a protein witha basic helix-loop-helix (bHLH) domain. To investigate whether this genemight be affected by mutations at the ind locus, we sequenced the codingregion in each of our mutant alleles. All five were found to containsingle nucleotide changes within the coding region, and three, includingind-2, would cause production of a truncated protein without the bHLHdomain. Complementation using a 3.4 kb genomic fragment spanningAt4g00120 rescues the ind mutant phenotype, further confirming that INDis the GT140 bHLH factor.

Analysis of IND cDNA clones derived from 5′ and 3′ RACE-PCR suggeststhat the IND transcript is 751 nucleotides (nt), with a 510 nt openreading frame, and 5′ and 3′ untranslated regions of 40 and 201 nt,respectively.

IND Represents a Unique Class of Eukaryotic bHLH Proteins

Transcriptional regulators with a bHLH domain bind DNA through residuesin the basic region while the helix-loop-helix domain promotesdimerization, allowing family members to form hetero- or homo-dimers(Murre, C., et al. Cell 56, 777-783 (1989)). Together, the two basicregions of the dimer usually recognize specific palindromic DNA hexamerswith the consensus sequence CANNTG, such that each bHLH protein binds ahalf-site. Eukaryotic bHLH proteins have been classified into six majorgroups according to their DNA-binding specificity, the presence ofadditional characteristic domains, and phylogenetic analysis (Ledent,V., and Vervoort, M., Genome Res. 11, 754-770 (2001)). All previouslycharacterized yeast and plant bHLH proteins have been assigned to theancestral B-class, which bind to the CACGTG E-box (also known as theplant G-box).

Comparison of the bHLH domain of IND with those of other eukaryoticfamily members has shown that the basic region is atypical. All A- andB-class bHLH proteins contain a critical glutamic acid residue (E) atsite 9 within the basic region. This particular residue has been shownto contact the outer CA nucleotides of the E-box and is required for DNAbinding (Fisher, F., and Goding, C. R. (1992). EMBO J. 11, 4103-4109).In the basic region of IND and several other closely related plantsequences, an alanine residue (A) is present at site 9 instead of theglutamic acid. Although certain C-class bHLH-PAS proteins such asSingle-minded (Sim) and Trachealess (Trh) also have atypical basicregions with alanines at this position, IND is not closely related tomembers of this class nor does it contain a predicted PAS domain (Nambu(1991) Cell 67:1157-1167; Wilk, R. (1996) Genes Dev. 10:93-102).

IND shares more than 60% sequence identity within the bHLH domain withat least twenty-seven other predicted plant bHLH proteins with atypicalbasic regions. However, sequence conservation between IND and the fiveclosest Arabidopsis relatives is primarily restricted to the bHLHdomain. For instance, the most related sequence, At5g09750, shares 82%identity with IND in the bHLH domain, but only 35% elsewhere.

ALC, which is also required for fruit dehiscence, shares only 42%identity with IND in the bHLH domain, and, like most Arabidopsis bHLHfamily members, shows characteristics of the B-class basic domain(Rajani, supra; Buck, M. J., and Atchley, W. R. J. Mol. Evol. 56,742-750 (2003)). The similarity between the alc-1, alc-2, and ind-1mutant phenotypes is intriguing, as alc-1 is predicted to be a nullallele (Rajani, supra), but ind-1 is not. Molecular analysis hasrevealed that the ind-1 mutation would result in substitution of aphenylalanine for a leucine within the first helix of the bHLH domain.This particular residue is observed in >98% of all known eukaryotic bHLHproteins, and has been shown from structural studies of the Maxhomodimer to pack together with other conserved hydrophobic amino acidsin the second helix and stabilize the intramolecular interactions of thefour-helix bundle (Ferré-D'Amaré, et al. Nature 363:38-45 (1993);Atchley, W. R., et al. J. Mol. Evol. 48:501-516 (1999); Atchley, et al.Mol. Biol. Evol. 17:164-178 (2000)). Substitution of another hydrophobicresidue at this position, although conservative, appears tosignificantly reduce activity of the ind-1 protein.

In contrast to ind-1, replacement of an arginine with a histidine in thebasic region of the ind-3 allele, results in a phenotypeindistinguishable from that of the ind-2 allele. As this arginine is oneof several residues known to make specific phosphate contacts within theDNA consensus sequence (Ferré-D'Amaré, et al. Nature 363:38-45 (1993);Ma, et al. Cell 77:451-459 (1993)), these results suggest thatdisruption of DNA binding is enough to abolish IND function entirely.

Expression of IND Expands Throughout the Valves of ful Fruit

To determine the pattern of IND expression in wild-type and mutantfruit, we performed antisense in situ hybridization with an IND-specificprobe. After fertilization, IND is expressed in stripes about four cellswide at the margins of developing wild-type fruit. We also detected INDexpression in the only valve layer which becomes lignified later infruit development, and in the vascular bundles of the replum. Like SHP1and SHP2 (Ferrándiz, et al. Science 289:436-438 (1999)), expression ofIND expands throughout the valves of ful mutant fruit, indicating thatFUL is required to restrict IND expression at the margin from thevalves.

Expanded IND Activity in ful Fruit Inhibits Growth and Causes EctopicLignification

Mutations in FUL cause severe defects in fruit growth, primarily due tolack of valve cell expansion after fertilization of the gynoecium (Gu,Q., et al. Development 125:1509-1517 (1998)). Previously, we have foundthat the ectopic expression of SHP1 and SHP2 in ful fruit does notaccount for their reduced growth, as shp1 shp2 ful fruit are notsignificantly longer than ful fruit (Ferrándiz, et al. Science289:436-438 (1999)). To determine whether ectopic IND activity couldinstead be primarily responsible for the expansion defects of ful fruit,we constructed the ind ful double mutant. Remarkably, we discovered thatfruit growth is considerably restored in ind ful fruit compared to fulfruit. Whereas mature ful fruit (2.5+/−0.2 mm) are 25% the length ofwild-type fruit (10.1+/−0.7), ind ful fruit (6.8+/−0.4) aresignificantly longer—more than twice the length of ful fruit and 67% thelength of wild-type. Scanning electron micrographs of ful and ind fulfruit compared to wild-type demonstrate the restoration of valveepidermal cell expansion due to loss of IND activity. Furthermore,differentiation of some epidermal cells into guard cells is seen in indful fruit, and is never observed in ful fruit.

In addition to growth defects, ful fruit also show ectopic lignificationof several valve cell layers (Ferrándiz, et al. Science 289:436-438(1999)). During wild-type fruit development, lignification of a singleinner valve cell layer is thought to contribute to fruit opening. In fulfruit, lignification of three additional valve layers occurs. Because wefound that IND is required for lignification of the wild-type fruitmargin, we expected that expanded IND activity might result in theectopic lignification of ful fruit. Indeed, as lignification of only theinner valve layer is observed in ind ful fruit, expanded IND activity isnot only largely responsible for the lack of valve expansion, but alsocauses the ectopic valve lignification of ful fruit.

Since SHP1, SHP2, and IND are each expressed at the margins of wild-typefruit, we have interpreted their expression throughout the valves of fulfruit as suggestive of an expansion of margin identity (Ferrándiz, etal. Science 289:436-438 (1999); this work). The notable suppression ofthe ful fruit phenotype conveyed by loss of ND activity providesexperimental validation of this hypothesis, and further supports a linkbetween the role of IND in promoting decreased cell expansion at themargin during fruit growth and its role in directing the laterlignification of a subset of margin cells. Furthermore, the phenotypicdifferences between ind ful and shp1 shp2 ful fruit constitutecompelling genetic evidence that IND expression and/or activity is notsimply regulated by SHP1 and SHP2.

An interesting lead to follow in the search for additional factors whichinhibit margin cell expansion, or promote their subsequentlignification, is YJ80. Like IND, expression of the YJ80 marker at themargin expands throughout the valves of ful fruit, and, with theexception of the few guard cells, is completely absent in the valves ofind ful fruit. As expected from analysis of YJ80 in shp1 shp2 and alcfruit, expression of YJ80 persists throughout the valves of shp1 shp2ful and alc ful fruit, strongly suggesting that the gene correspondingto this marker is specifically regulated by IND.

Plants with Ectopic IND Expression Produce ful-Like Fruit

To further explore the developmental effects of ectopic IND activity, wegenerated transgenic plants expressing IND under control of either theconstitutive cauliflower mosaic virus 35S promotor or the FUL promoter,which directs expression in the inflorescence meristem, cauline leaves,and throughout the developing valves of the gynoecium. Phenotypicanalysis revealed that 17 of 101 35S::IND and 48 of 135 FUL::IND T1plants produced ful-like fruit with severe growth defects. Furthermore,a significant number of 35S::IND and FUL::IND T1 plants exhibitedweakerful-like fruit phenotypes, much like the fruit produced by plantsconstitutively expressing SHP1 and SHP2. These results correspond wellwith our discovery that mutations in IND significantly suppress the fulfruit phenotype, and demonstrate that ectopic IND activity is sufficientto inhibit fruit growth.

Loss of IND, SHP, and ALC Activity Largely Suppresses the ful FruitPhenotype

Since mutations in IND not only have the most severe effect on margindevelopment, but also suppress the ful phenotype more dramatically thanmutations in ALC, or SHP1 and SHP2, we wondered if these transcriptionfactors regulate any aspects of margin development independently of IND.To address this question, we have conducted systematic genetic analysisto uncover the relative contributions of IND, SHP1, SHP2, and ALC tomargin development and to determine the extent their ectopic activitieshave on the ful fruit phenotype.

By comparing ind shp1 shp2, ind alc, and ind fruit, we observed anenhanced loss apical margin definition in ind shp1 shp2 fruit comparedto ind fruit, but did not detect any morphological differences betweenind alc and ind fruit. A smaller, but similar loss of margin definitionwas also evident in our examination of shp1 shp2 alc fruit compared toshp1 shp2 fruit. These results suggest that SHP1 and SHP2 do regulatesome aspects of margin development independently of IND and ALC, andthat ALC activity is primarily encompassed by IND.

The IND-independent activity of SHP1 and SHP2 is much more apparent whencomparing ind shp1 shp2 ful to ind ful fruit. Fruit length in ind shp1shp2 ful fruit (8.5+/−0.8 mm) is largely restored (84%) to wild-type,and the overall appearance of the fruit, while rumpled, is more likewild-type, due to increased lateral valve cell expansion. Furthermore,scanning electron micrographs of ind shp1 shp2 ful fruit compared towild-type, ful and ind ful fruit demonstrate the extensive restorationof guard cell differentiation due to loss of ectopic IND, SHP1, and SHP2activity. Support for the ALC-independent activity of SHP1 and SHP2 isalso more evident in comparing shp1 shp2 alc ful to alc ful fruit.Although the fruit length (5.1+/−0.4 mm) of shp1 shp2 alc ful fruit isonly partially restored (51%) compared to wild-type, it is significantlylonger than that of alc ful fruit (4.0+/−0.3 mm). Taken together, theseresults clearly indicate that SHP1 and SHP2 regulate factors involved inmargin development and cell expansion independently of IND and ALC.

Although our initial observations of ind alc fruit suggested that ALCmight not play any roles in margin development independent of IND,analysis of ind alc ful and ind alc shp1 shp2 ful fruit has revealedthat ALC does possess both IND- and SHP-independent roles. Fruitproduced by the ind alc ful mutant are significantly longer (8.2+/−0.6mm) than ind ful fruit (6.8+/−0.4 mm). Furthermore, while notsignificant, a slight increase in length is also observed in comparingind shp1 shp2 ful (8.5+/−0.8 mm) to ind alc shp1 shp2 ful (9.1+/−0.9 mm)fruit.

IND, SHP, ALC, and FUL Activities Contribute to Differentiation of theLignified Valve Layer

In addition to finding that SHP and ALC have IND-independent roles inmargin development, we also discovered that together with IND and FULthese factors are involved in specifying lignification of the lignifiedvalve layer. Examination of ind a/c shp1 shp2 fruit compared to wildtype revealed that a few cells in the lignified valve layer adjacent toeach valve margin fail to lignify. A similar, but less penetrant,retraction of lignified valve layer cells from the replum was alsoobserved in ind shp1 shp2 fruit. The appearance and size of thesenon-lignified cells is most like those found in the neighboringmesophyll cell layers.

In wild-type fruit, FUL is expressed throughout the valves (Gu et al.,1998). Previously we have found that the expression of FUL retractsslightly from the valve margin in shp1 shp2 mutant fruit (Ferrándiz etal., 2000b). In ind mutants, we also observe a slight retraction of theFUL from the margin. The retraction of FUL from the margin is moredramatic in ind a/c shp1 shp2 quadruple mutant fruit, and correlateswith the absence of lignified cells near the margin in the lignifiedvalve layer. When FUL activity is removed in the ind a/c shp1 shp2 fulquintuple mutant, lignification of the lignified valve layer iscompletely absent except for a few cells at the base of the fruit. Theobservation that lignification of this layer is reduced but noteliminated in ind shp1 shp2 ful quadruple mutant fruit (data not shown)indicates that ALC also plays a role in specifying this cell type. Sincethe lignified valve layer is completely eliminated only when all fivetranscription factors—IND, ALC, SHP1, SHP2, and FUL—are inactivated, itis evident that each factor contributes to lignification of this layer.

Experimental Procedures

Plants

Mutant alleles of IND and ALC were obtained through ethylmethanesulphonate mutagenesis as previously described (Liljegren, Nature404:766-770 (2000)). The ind-2 allele contains a single nucleotidedeletion within codon 26, which results in a frameshift and productionof a truncated protein of 35 amino acids. The ind-1 and ind-3 allelescontain nucleotide substitutions within codons 112 and 99, which changea leucine to a phenylalanine and an arginine to a histidine,respectively. The ind-4 and ind-5 alleles contains nucleotidesubstitutions within codons 63 and 13, which change a glutamine and atryptophan to stop codons, causing production of truncated proteins of62 and 12 amino acids, respectively. The alc-2 mutation contains anucleotide substitution at the splice donor site of the third intron,which should disrupt splicing of the transcript region encoding thesecond helix of the bHLH domain. The ind-2 and alc-2 alleles werebackcrossed three times to Ler and used for subsequent genetic analyses,along with the shp1-1, shp2-1, and ful-5 alleles.

Plants homozygous for the ind-2 and/or alc-2 alleles were detected withCAPS (cleaved amplified polymorphic sequence) markers based on an AluIsite abolished by the ind-2 mutation and an AseI site introduced by thealc-2 mutation. The shp1-1 and shp2-1 mutations were detected asdescribed previously.

cDNA Analysis

To examine the transcripts produced at the IND locus, 5′ and 3′ RACE-PCR(Roche) were performed as described by the manufacturer using total orpolyA RNA, respectively, as template. For 5′ RACE,5′-GAGTTGTGGTAATAACAAAGGTAAG-3′ was used in the reverse transcriptasereaction, and additional nested oligos 5′-GGCTTCGTCGAGCATGGAAGC-3′ and5′-GAGCAACCACCGTCTGAGGATCG-3′ were used in subsequent rounds of PCR. For3′ RACE, oligo dT was used in the reverse transcriptase reaction, andthe nested primer 5′-CCCTGCCACGGTCCCTAAGC-3′ in a subsequent round ofPCR. The resulting fragments were cloned into pCR2.1 (Invitrogen) andsequenced. Analysis of IND cDNA clones derived from 5′ and 3′ RACE-PCRsuggests that the IND transcript is 751 nucleotides (nt), with a 510 ntopen reading frame, and 5′ and 3′ untranslated regions of 40 and 201 nt,respectively. Further support for the assigned open reading frame isprovided by an IND EST (AF488578).

Marker Analyses

To isolate flanking sequence from the GT140 marker (Sundaresan, et al.(1995) Genes Dev. 9:1797-1810), TAIL (Thermal Asymmetric Interlaced)/PCRwas performed using nested oligos specific for the left and righttransposon borders and degenerate primers as described previously(Tsugeki, et al. (1996) Plant J. 10:479-489). The transposon insertionwas detected 2782 nucleotides 5′ of the predicted ATG of At4g00120, andcreates a duplication of 8 bp (GTATTTGC) flanking the insertion site.

The YJ80 enhancer trap line was generated by Agrobacterium-mediatedtransformation with the plasmid pOCA-28-15-991. Transgenic plantscontaining YJ80, GT140, YJ36 or a FUL marker were crossed into mutantplants. For β-glucuronidase expression analyses, fruit from wild-typeand mutant plants were fixed, sectioned and stained with minormodifications.

Generation of Transgenic Plants

Using genomic DNA from the GT140 insertion line as a template, a 2.9 kbregion spanning from 180 nucleotides upstream of the predicted At4g00120translational start site and extending into the Ds insertion element wasPCR amplified. This fragment was cloned into pCR2.1 (Invitrogen), thenexcised as a SalI/BamHI fragment and cloned into the planttransformation vector, pBI101.3. 17 of 38 transgenic Ti lines producedindehiscent fruit.

A 3.4 kb genomic region of IND, extending 2740 bases 5′ and 480 bases 3′of the coding region, was PCR amplified using Columbia DNA as atemplate. This fragment was cloned into pCR2.1, then excised as an XbaIfragment and cloned into the pEL112 plant transformation vector.Basta-resistant transgenic plants exhibiting a complemented phenotypewere PCR analyzed to confirm that they were homozygous for the ind-2allele.

A full-length IND cDNA was PCR amplified with the oligos(5′-CGTCGACGATGAAAATGGAAAATGGTATGTATA-3′ and5′-CGGATCCGTTCATCAGGGTTGGGAGTTGTG-3′) using Columbia DNA as a template.After cloning this product into pCR2.1, a SalI/BamHI fragment containingthe IND cDNA was cloned into the pBIN-JIT vector. The resultingconstruct placed IND under the control of a tandem repeat of the 35Spromoter.

Microscopy and Histology

Wild-type (Landsberg erecta ecotype), mutant, and transgenic fruit andflowers were fixed, prepared, and analyzed by scanning electronmicroscopy as previously described. Tissue fixation and phloroglucinolstaining of paraplast sections (8 or 10 μm) from late stage 17 fruitwere done as described (Liljegren et al., 2000). Plastic sections (3 μm)were prepared with JB4 resin (Electron Microscopy Sciences) as described(Roeder et al., 2003) from the tenth stage 17 fruit on wild-type andmutant inflorescences.

In Situ Hybridization

Wild type and mutant sections were hybridized with antisense or senseRNA as described. The IND probe was synthesized with T7 RNA polymerasefrom a SalI-digested pINDAS template to generate a 328 nucleotideantisense transcript encompassing the 5′ region through part of thefirst helix of the bHLH domain. pINDAS was created by ligating the INDproduct PCR amplified from Colombia DNA with5′-GAGCAACCACCGTCTGAGGATCG-3′ and5′-CGTCGACGATGAAAATGGAAAATGGTATGTATA-3′ into the pCR2.1 vector.

Example 2

Two IND1 orthologs were isolated from Brassica napus plants. SinceBrassica napus has an allotetraploid genome, it is not surprising thattwo different IND1 orthologs are present in the genome. The twosequences are designated Bn IND1 and Bn IND2. An alignment of the aminoacid sequences of Bn IND1 and Bn IND2 with SEQ ID NO:2 are depicted inFIG. 1. An alignment of the nucleotide sequences of Bn IND1 and Bn IND2with SEQ ID NO:1 are depicted in FIG. 2. The amino acid sequence of BnIND1 is approximately 63% identical to SEQ ID NO:2 of the presentinvention, as measured with BLAST without the low complexity filter. Theamino acid sequence of Bn IND2 is approximately 67% identical to SEQ IDNO:2 of the present invention. Like the Arabidopsis IND1 sequence, theBrassica IND sequences include an alanine residue (A) at site 9 of thebasic region instead of the glutamic acid (e.g., position 140 of SEQ IDNO:9 and position 112 of SEQ ID NO:10).

Transformation of either Bn IND1 or Bn IND2 into ind1 mutant Arabidopsisplants resulted in complementation of the mutant phenotype. Theseresults demonstrate that Bn IND1 and Bn IND2 carry out the same basicfunctions as IND1.

Example 3

To examine the effect of ectopic expression of IND1 on lignification, weintroduced a 35S::IND1 construct into Arabidopsis plants and assayed forlignification. Upon germination, Arabidopsis plants produce a rosette ofleaves on the surface of the soil. These leaves are closely spaced as aresult of the lack of internode elongation between leaves. Upon thetransition to reproductive development in Arabidopsis, the main stem isoften referred to as the inflorescence stem, since it is responsible forproducing flowers on its flanks. This stem elongates considerably,giving the plant its characteristic height. Inspection of thelignification patterns in the inflorescence stem of wild-type plants,determined by the lignin-specific phloroglucinol stain of a stemsection, revealed the normal pattern of stem lignification in thetracheary elements. A similar stem section from 35S::IND1 plant stemsappeared to reveal ectopic lignification. The 35S::IND1 plants were moreextensively lignified than wild-type plants, indicating that the ectopicexpression of IND1 in the stem is sufficient to promote ectopiclignification of stem cells.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. An isolated nucleic acid comprising a polynucleotide encoding apolypeptide at least 90% identical to SEQ ID NO:9 or SEQ ID NO:10. 2.The isolated nucleic acid of claim 1, wherein the polypeptide comprisesSEQ ID NO:9.
 3. The isolated nucleic acid of claim 1, wherein thepolypeptide comprises SEQ ID NO:10.
 4. The isolated nucleic acid ofclaim 1, wherein the polynucleotide comprises SEQ ID NO:7.
 5. Theisolated nucleic acid of claim 1, wherein the polynucleotide comprisesSEQ ID NO:8.
 6. The isolated nucleic acid of claim 1, wherein thepolynucleotide comprises SEQ ID NO:11.
 7. The isolated nucleic acid ofclaim 1, wherein the polynucleotide comprises SEQ ID NO:12.
 8. Anexpression cassette comprising a promoter operably linked to apolynucleotide, or a complement thereof, wherein the polynucleotide isat least 90% identical to at least 200 contiguous nucleotides of SEQ IDNO:7 or SEQ ID NO:8.
 9. The expression cassette of claim 8, wherein thesequence comprises SEQ ID NO:9.
 10. The expression cassette of claim 8,wherein the sequence comprises SEQ ID NO:10.
 11. The expression cassetteof claim 8, wherein the polynucleotide is at least 90% identical to anucleotide sequence encoding SEQ ID NO:9.
 12. The expression cassette ofclaim 8, wherein the polynucleotide comprises a nucleotide sequenceencoding SEQ ID NO:9.
 13. The expression cassette of claim 8, whereinthe polynucleotide is at least 90% identical to a nucleotide sequenceencoding SEQ ID NO:10.
 14. The expression cassette of claim 8, whereinthe polynucleotide comprises a nucleotide sequence encoding SEQ IDNO:10.
 15. The expression cassette of claim 8, wherein the promoter isconstitutive.
 16. The expression cassette of claim 8, wherein thepromoter is tissue specific.
 17. The expression cassette of claim 8,wherein the promoter is a dehiscence zone specific promoter.
 18. Theexpression cassette of claim 8, wherein the polynucleotide is operablylinked to the promoter in the antisense orientation.
 19. The expressioncassette of claim 8, wherein the polynucleotide is operably linked tothe promoter in the sense orientation.
 20. A plant comprising arecombinant expression cassette, the expression cassette comprising apromoter operably linked to a polynucleotide, or a complement thereof,wherein the polynucleotide is at least 90% identical to at least 200contiguous nucleotides of a sequence encoding SEQ ID NO:9 or SEQ IDNO:10.
 21. The plant of claim 20, wherein the sequence comprises SEQ IDNO:9.
 22. The plant of claim 20, wherein the sequence comprises SEQ IDNO:10.
 23. The plant of claim 20, wherein the polynucleotide is at least90% identical to a nucleotide sequence encoding SEQ ID NO:9.
 24. Theplant of claim 20, wherein the polynucleotide comprises a nucleotidesequence encoding SEQ ID NO:9.
 25. The plant of claim 20, wherein thepolynucleotide is at least 90% identical to a nucleotide sequenceencoding SEQ ID NO:10.
 26. The plant of claim 20, wherein thepolynucleotide comprises a nucleotide sequence encoding SEQ ID NO:10.27. The plant of claim 20, wherein the promoter is constitutive.
 28. Theplant of claim 20, wherein the promoter is tissue specific.
 29. Theplant of claim 20, wherein the promoter is a dehiscence zone specificpromoter.
 30. The plant of claim 20, wherein the polynucleotide isoperably linked to the promoter in the antisense orientation.
 31. Theplant of claim 20, wherein the polynucleotide is operably linked to thepromoter in the sense orientation.
 32. The plant of claim 20, whereinlignification is reduced in valve margin cells of the plant.
 33. Theplant of claim 20, wherein lignification is enhanced in the plant. 34.The plant of claim 20, wherein the plant is a Brassica species.
 35. Theplant of claim 20, wherein the plant is characterized by delayed seeddehiscence compared to a plant not comprising the expression cassette.36. A method of delaying fruit dehiscence in a plant, the methodcomprising, suppressing expression of an IND1 nucleic acid in the plantby introducing into the plant a recombinant expression cassettecomprising a promoter operably linked to a polynucleotide, or acomplement thereof, wherein the polynucleotide is at least 90% identicalto at least 200 contiguous nucleotides of a sequence encoding SEQ IDNO:9 or SEQ ID NO:10; and selecting a plant with delayed fruitdehiscence compared to a plant in which the expression cassette has notbeen introduced.
 37. The method of claim 36, wherein the sequencecomprises SEQ ID NO:9.
 38. The method of claim 36, wherein the sequencecomprises SEQ ID NO:10.
 39. The method of claim 36, wherein thepolynucleotide is at least 90% identical to a nucleotide sequenceencoding SEQ ID NO:9.
 40. The method of claim 36, wherein thepolynucleotide comprises a nucleotide sequence encoding SEQ ID NO:9. 41.The method of claim 36, wherein the polynucleotide is at least 90%identical to a nucleotide sequence encoding SEQ ID NO:10.
 42. The methodof claim 36, wherein the polynucleotide comprises a nucleotide sequenceencoding SEQ ID NO:10.
 43. The method of claim 36, wherein the promoteris constitutive.
 44. The method of claim 36, wherein the promoter istissue specific.
 45. The method of claim 36, wherein the promoter is adehiscence zone specific promoter.
 46. The method of claim 36, whereinthe polynucleotide is operably linked to the promoter in the antisenseorientation.
 47. The method of claim 36, wherein the polynucleotide isoperably linked to the promoter in the sense orientation.
 48. The methodof claim 36, wherein the recombinant expression cassette is introducedinto the plant using Agrobacterium.
 49. The method of claim 36, whereinthe plant is a Brassica species.